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WO2025186424A1 - Procédés pour technique de coupe et de marquage (cut&tag) - Google Patents

Procédés pour technique de coupe et de marquage (cut&tag)

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Publication number
WO2025186424A1
WO2025186424A1 PCT/EP2025/056228 EP2025056228W WO2025186424A1 WO 2025186424 A1 WO2025186424 A1 WO 2025186424A1 EP 2025056228 W EP2025056228 W EP 2025056228W WO 2025186424 A1 WO2025186424 A1 WO 2025186424A1
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WIPO (PCT)
Prior art keywords
chromatin
cell
dna
antibody
cells
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PCT/EP2025/056228
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WO2025186424A8 (fr
Inventor
Nathalie Allegra Faye SONDERMANN
Irina Panteleeva
Damien Jean-Pol Bernard Ghislain CALAY
Céline SABATEL
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Diagenode SA
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Diagenode SA
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Priority claimed from EP24162157.2A external-priority patent/EP4613870A1/fr
Application filed by Diagenode SA filed Critical Diagenode SA
Publication of WO2025186424A1 publication Critical patent/WO2025186424A1/fr
Publication of WO2025186424A8 publication Critical patent/WO2025186424A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the disclosure relates to improved methods that profile chromatin-associated proteins for their chromatin binding sites.
  • the methods described herein allow for reliable analysis of a broad range of cell numbers and/or protein-chromatin interactions.
  • ChIP chromatin immunoprecipitation
  • the harvested cells are cross-linked with formaldehyde to ensure covalent, reversible cross-linking of the proteins with the DNA.
  • the cells are lysed and the chromatin is sheared off by sonication.
  • Antibodies are added that bind against the chromatin- associated protein of interest as well as to magnetic beads coated with protein A, allowing the chromatin-bound fraction being retained during washing steps, while the non-target chromatin portion can be removed without centrifugation.
  • the immunoprecipitated DNA is then eluted from the beads with SDS-containing buffer, the cross-linking is relieved by heating to 65°C in the presence of SDS and sodium chloride.
  • the recovered DNA is purified and used for enrichment testing by qPCR, library preparation and sequencing.
  • ChIP requires a high number of cells as starting material, typically 10 6 to 10 7 cells, and a low starting quantity correlates with lower data quality.
  • harsh crosslinking conditions can mask the epitope, resulting in loss of signal, and chromatin that does not become soluble after shearing is lost in the process.
  • chromatin preparation is time consuming, has a high variability between batches of sheared chromatin, and results in high sequencing background.
  • CUT&Tag Cleavage Under Targets and Tagmentation
  • cells are bound to magnetic concanavalin A-coated beads during the process up to DNA extraction and purification.
  • An antibody against the desired chromatin mark enters the permeabilized cells and marks the site in the genome.
  • a subsequently added fusion protein consisting of protein A and transposase Tn5 is bound to the antibody. After binding of the protein A part to the antibody and activation of Tn5, the latter cuts the DNA and incorporates sequencing adapters at the site. These can be sequenced after extraction and PCR amplification.
  • CUT&Tag can successfully detect abundant proteins with strong interactions with DNA, such as histone proteins.
  • proteins that are less abundant or have a weaker, more dynamic interaction with DNA and therefore detach more easily from DNA require a higher number of cell inputs and cannot be reliably detected with CUT&Tag.
  • CUT&Tag usually involves the use of high salt concentrations to remove unbound pA-Tn5 during the washing steps which is necessary to decrease off-target tagmentation, proteins tightly bound to DNA may remain bound to their target site in chromatin, while weaker interacting proteins could be removed under these harsh washing conditions.
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprising:
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule;
  • the transposome comprises a transposase and a first and second DNA molecule.
  • the first DNA molecule may comprise a first transposase recognition site and the second DNA molecule may comprise a second transposase recognition site.
  • At least the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in presence of a Good’s buffer.
  • the Good’s buffer is selected from Tricine, MES, ADA, PIPEs, ACES, MPOSO, Cholamine chloride, MOPS, BES, TES, DIPSO, TASO, Acetamidoglycine, POPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tris, Glycinamide, Glycylglycine, Bicine and TAPS, optionally wherein the Good’s buffer is Tricine.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed at a salt concentration of ⁇ 300 mM NaCI.
  • the cells prior to the permeabilization step are subjected to mild cross-linking conditions.
  • the mild cross-linking conditions are 0.1% formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min.
  • the mild cross-linking conditions are 0.1% (v/v) formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min.
  • MgCI 2 is not added prior to activating the transposase.
  • the chromatin-associated factor of interest is a non-histone protein, optionally a transcription factor or a histone reader protein.
  • the number of cells is ⁇ 100,000, optionally ⁇ 50,000, ⁇ 10,000, ⁇ 5,000 or ⁇ 1,000.
  • the cell is permeabilized using Triton-X-100, optionally at a concentration of 0.1 %.
  • the crowding agent is selected from a group comprising sucrose, hexylene glycol and glycerol, optionally wherein the crowding agent is sucrose.
  • the cells prior to the permeabilization step, are bound to beads, optionally magnetic beads.
  • all method steps are performed using total buffer volumes of ⁇ 200 pl.
  • the amount of transposase that is linked to a specific binding agent is ⁇ 1:250 of the reaction volume.
  • the dilution of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 1:250.
  • the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 15 ng/pl.
  • the amount of first and/or second antibody per reaction is
  • Another embodiment comprises performing steps (a) to (f) in a first replicate and the following steps in a second replicate: purifying DNA from the cell, contacting the purified DNA with the transposome; activating the transposase; determining the sequence of the excised and tagged DNA in the second replicate; wherein the sequence of the excised and tagged DNA segment determined in the second replicate is used as a normalization control in the step of determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell determined in step (f) in the first replicate, wherein optionally at least the steps of contacting the purified DNA with the transposome and activating the transposase are performed in presence of a crowding agent.
  • an input sample is set aside prior to the permeabilization step and subjected to method steps (d) to (f), and wherein the input sample is used for normalization in the step of determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell based on the determined sequence of the excised and tagged DNA.
  • an antibody targeting the chromatin-associated factor of interest is used as first antibody and for a second portion of the cell population IgG is used as first antibody, and wherein the sequence of the excised DNA determined for the portion of the cell population using IgG as first antibody is used for normalization in the step of determining the sequence of the excised DNA for the portion of cell population using the antibody targeting the chromatin-associated factor of interest as first antibody.
  • Another aspect relates to a method for generating a normalization control suitable for use in the methods described above, comprising the following steps: purifying DNA from the cell; contacting the purified DNA with a transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; and determining the sequence of the excised and tagged DNA
  • Another aspect relates to a method for generating a normalization control suitable for use in the methods described above, comprising the following steps: purifying DNA from the cell; contacting the purified DNA with a transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; and determining the sequence of the excised and tagged DNA; wherein the steps of contacting the purified DNA with the transposome and activating the transposase are performed in presence of a crowding agent.
  • a method for assessing the quality of the methods described above by determining the percentage of false positives resulting from off-target transposase activity comprises the following steps: (i) conducting steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell at least twice,
  • step (iii) identifying a subset of the chromatin binding sites identified in step (ii) that does not overlap with a reference set of chromatin binding sites determined for the chromatin-associated factor of interest identified using ChlP-seq,
  • step (iv) identifying a subset of the subset of chromatin binding sites identified in step (iii) that overlaps with a reference set of chromatin sites identified using ATAC-seq;
  • step (v) determining the number of chromatin binding sites identified in step (iv) and dividing said number with the number of chromatin binding sites identified in step (ii) in order to obtain the percentage of false positives resulting from off-target transposase activity.
  • conducting steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin- associated factor of interest in a cell at least twice comprises conducting steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin- associated factor of interest in a first replicate and repeating steps (a) to (f) of the above described method for determining at least one chromatin binding site of a chromatin-associated factor of interest in at least a second replicate.
  • conducting steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin- associated factor of interest in a cell at least twice comprises conducting steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin- associated factor of interest in a first replicate and repeating steps (a) to (f) of the above described methods for determining at least one chromatin binding site of a chromatin- associated factor of interest at least once in the same replicate.
  • the chromatin binding site of the chromatin-associated factor of interest is present in closed chromatin or heterochromatin.
  • the first antibody is an antibody against H3K27me3 or H3K9me3.
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprises the following steps:
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule
  • the purified DNA in the second replicate is not contacted with the first antibody that specifically binds the chromatin-associated factor of interest and with the second antibody that specifically binds to the first antibody.
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprising:
  • permeabilizing the cell with a non-saponin permeabilization reagent, optionally selected from Triton X-100 and Tween-20.
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule;
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprising:
  • permeabilizing the cell with a non-saponin permeabilization reagent, optionally selected from Triton X-100 and Tween-20.
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule;
  • FIG. 1 Concentration of sequencing libraries obtained with the conventional CUT&Tag protocol using 50,000 HPC7 wild-type (WT) or Phf19 knockdown (kd) cells and primary antibodies against SLIZ12, JARID2 and PHF19. IgG was used as control. The bars represent the mean of library concentration ⁇ SD of duplicates.
  • FIG. 2 Genomic tracks at the Nanog gene locus showing data obtained with the conventional CUT&Tag protocol in 50,000 wild-type (WT) or Phf19 knockdown (kd) HPC7 cells using primary antibodies against SUZ12, JARID2 and PHF19.
  • FIG. 3 Concentration of sequencing libraries obtained with the conventional CUT&Tag protocol using 50,000 or 300,000 K-562 cells and primary antibodies against H3K4me3, H3K27me3, H3K9me3, CTCF, NRF1 and HDAC2. IgG was used as control. The bars represent the mean of library concentration ⁇ SD of duplicates.
  • Figure 4 Genome tracks showing CTCF, HDAC2 and NRF1 obtained with 50,000 or 300,000 K-562 cells using the conventional CUT&Tag protocol or 4 million K-562 cells using ChlP.
  • CTCF samples at the H19 gene CTCF samples at the H19 gene.
  • HDAC2 samples at the Spate'll and Lss genes HDAC2 samples at the Spate'll and Lss genes.
  • FIG. 5 Genome tracks of CUT&Tag experiments on H3K4me3 using Triton X-100 (TX) and digitonin (Dig) for cell permeabilisation at the genomic locus of Gapdh and Phf19. CUT&Tag experiments were performed using 50,000 K-562 cells. For comparison, ChlP-seq data with 50,000 K-562 cells are shown.
  • FIG. 6 Concentration of sequencing libraries obtained with the improved CUT&Tag protocol with or without crosslinking and with the conventional CUT&Tag protocol using 50,000 K-562 cells and targeting CTCF, H3K3me3 or IgG (control). The bars represent the mean of library concentration ⁇ SD of duplicates.
  • Figure 7 Concentration of sequencing libraries obtained with the improved CUT&Tag protocol targeting CTCF, EZH2, HDAC2, H3K27me3 and IgG using either 100,000, 50,000, 10,000 or 5,000 K-562 cells. Bars represent library concentration in mean ⁇ SD of duplicates.
  • Figure 8 Peak correlation of CUT&Tag samples assessing CTCF, EZH2, HDAC2 using 50,000 or 100,000 cells (duplicates) and compared to ENCODE ChlP-seq data assessing the same chromatin-binding proteins. CUT&Tag samples obtained with the protocol disclosed herein are highly correlated with each other based on binding site occupancy, but not with ENCODE ChlP-seq data.
  • Figure 9 Heatmaps of (A) CTCF, (B) EZH2 and (C) HDAC2 CUT&Tag samples generated using the improved protocol and ENCODE ChlP-seq samples 3 kb upstream and downstream of the TSS. Despite their heterogeneity, most of the CUT&Tag samples show a higher signal than the ENCODE ChIP samples.
  • FIG. 10 Genome tracks of CTCF, EZH2 and HDAC2 CUT&Tag samples and their respective ENCODE ChlP-seq reference data.
  • CTCF H19 locus.
  • EZH2 HoxA locus and Phf19 locus.
  • HDAC2 Btn3a3 locus.
  • Figure 11 (A) Concentration of sequencing libraries normalised for 14 PCR cycles to be comparable obtained with the improved CUT&Tag protocol assessing H3K27me3, CTCF, EZH2, SUZ12 and IgG (control) using 50,000 cells in WT and SUZ12 _/ - mESCs. Bars represent library concentration in mean ⁇ SD of duplicates. (B) Enrichment of H3K27me3, CTCF, EZH2 and SUZ12 on target loci relative to negative control loci. IgG was used as control. Bars represent relative enrichment in mean ⁇ SD of duplicates.
  • Figure 12 CUT&Tag for CTCF in WT and Suz12 _/ - mESCs as Suz12 KO-independent control.
  • A Overlap of gene sets;
  • B Functional analysis using Enrichr;
  • C Genome tracks of CTCF at the HoxA cluster.
  • Figure 13 CUT&Tag for H3K27me3, EZH2, SUZ12 in WT and Suz12- Z - mESCs.
  • A Common and specific gene sets between H3K27me3 duplicates, EZH2 triplicates and SUZ12 triplicates, respectively;
  • B Genome tracks of one replicate for H3K27me3, EZH2 and SUZ12 in WT and Suz12 _/ - mESCs using the same respective scale;
  • C Common and specific genes between the respective WT and Suz12 _/ - samples for H3K27me3, EZH2 and SUZ12;
  • D Overlap of gene sets between one replicate of H3K27me3, EZH2 and SUZ12.
  • Figure 14 Comparison of SUZ12 CUT&Tag in WT and Suz12 _/ - cells at peak level.
  • A Number of common and specific peaks between WT and Suz12 _/ -;
  • B Signal intensity at peaks;
  • C Genomic distribution of peaks.
  • Figure 15 Characteristics of WT-only, common and KO-only genes identified using SUZ12 CUT&Tag in WT and Suz12 _/ - cells.
  • A Overlap of the genes underlying peaks identified in SUZ12 CUT&Tag in WT and Suz12 _/ - cells.
  • B Functional analysis of the gene subsets.
  • C Count of reads at TSS of gene subsets identified by SUZ12 CUT&Tag in WT and Suz12 _/ - samples.
  • FIG. 16 (A) Concentration of sequencing libraries obtained with the improved CUT&Tag protocol targeting H3K27me3 using two different antibodies (Diagenode, D; Merck Millipore; M), CTCT and SUZ12 using 50,000 mESCs. IgG was used as control. Bars represent library concentration in mean ⁇ SD of duplicates. (B) Enrichment of H3K27me3, CTCF and SUZ12 on target loci relative to negative control loci. IgG was used as control. Bars represent relative enrichment in mean ⁇ SD of duplicates.
  • Figure 17 Peak correlation of CUT&Tag samples assessing H3K27me3 using two different antibodies (Diagenode, D; Merck Millipore; M) using 50,000 SUZ12 WT mESCs. Normalisation with either IgG or input increases correlation among H3K27me3 replicates.
  • Figure 18 Peak correlation of CUT&Tag samples assessing SUZ12 using 50,000 SUZ12 WT mESCs. Normalisation with either IgG or input.
  • FIG. 19 Genome tracks of CUT&Tag in WT and Suz12 _/ - mESCs.
  • A H3K27me3 CUT&Tag in WT and Suz12 _/ - mESCs.
  • SUZ12 ChIP red
  • IgG and input tracks are shown as controls. Normalization with IgG or input (black) leads to the disappearance of the H3K27me3 peaks in the KO sample (circled in red), while the broad peak called at the HoxA cluster in the H3K27me3 WT sample (green) remains;
  • B CTCF CUT&Tag in WT and Suz12 _/ - mESCs.
  • CTCF ChlP-seq data from ENCODE are shown as controls.
  • Figure 20 Percentage of peaks not overlapping with reference SUZ12 ChlP-seq data (NOC) and percentage of peaks not overlapping with reference SUZ12 ChlP-seq peaks but with ATAC-seq peaks (NOCOA) of SUZ12 CUT&Tag without normalisation or upon normalisation with IgG and input, respectively. Bars represent mean of duplicates.
  • Figure 21 Genome tracks of CUT&Tag samples assessing H3K27me3 and SUZ12 at the HoxA cluster in 50,000 WT and Suz12 _/ - mESCs. Peaks were IgG-normalised.
  • FIG. 22 The comparison of CUT&Tag in WT and Suz12 _/ - mESCs shows the successful application of the CUT&Tag protocol described herein and a large overlap of peaks in the M H3K27me3 replicates.
  • A Metaplots of CUT&Tag samples using antibodies against H3K27me3 by Diagenode and Merck Millipore, respectively, or against SUZ12 in WT and Suz12 _/ - mESCs, respectively, as well as obtained with IgG in the same cells.
  • B Overlap of IgG-normalised peaks between H3K27me3 replicates obtained using the Merck Millipore (M) antibody.
  • C Signal at sites of common and specific peaks for H3K27me3 replicates obtained using the Merck Millipore (M) antibody.
  • Figure 23 Normalisation decreases detected peaks and genes in Suz12 _/ - cells and reduces the overlap with WT data sets.
  • A Overlap of H3K27me3 (Merck Millipore, M) peaks obtained by CUT&Tag in WT and Suz12 _/ - cells upon normalization with IgG (left) and without normalization (right).
  • B ATAC-seq signal in IgG-normalised H3K27me3 (Merck Millipore, M) WT and Suz12 _/ - peak sets.
  • Figure 25 CUT&Tag protocol described herein achieves higher library concentrations and enrichment at target loci compared to previous protocol versions.
  • A Concentration of sequencing libraries obtained with the improved CUT&Tag protocol targeting H3K27me3, SUZ12 and JARID2 using 5,000 mESCs. IgG and input were used as controls. Bars represent library concentration in mean ⁇ SD of duplicates.
  • B Enrichment of H3K27me3, SUZ12 and JARID2 on target loci relative to negative control loci. IgG was used as control. Bars represent relative enrichment in mean ⁇ SD of duplicates.
  • Figure 26 Genome tracks obtained with the improved CUT&Tag protocol targeting H3K27me3, SUZ12 and JARID2 using 5,000 mESCs showing four different loci: (A) Cbx2,4,8 (left), HoxA cluster (right). (B) Fsd2 (left), Mir9-3hg (right). ChlP-Seq data (gold), and IgG and H3K27me3 CUT&Tag data obtained using 50,000 mESCs are shown as controls.
  • Figure 27 Functional analysis of CUT&Tag assessing H3K27me3 (A), SUZ12 (B) and JARID2 (C) in 5,000 mESCs using Enrichr.
  • Figure 28 Number of peaks obtained for H3K27me3 in WT and SUZ12 _/ - mESCs with different peak callers and/or settings without normalization and after normalization with IgG, respectively.
  • Figure 29 NOCOA proportions obtained for H3K27me3 in WT and SUZ12 _/ - mESCs with different peak callers and/or settings without normalization and after normalization with IgG, respectively.
  • Figure 30 Genome tracks and peaks of MTF2 in WT-like mES cells (DMSO, green) and mESCs with an MTF2-KO (dTAG, red) using standard settings of peak callers epic2 and MACS2 at the HoxA cluster (right) and Alx1 gene (left). Blue: IgG samples. (A) without normalisation. (B) with IgG normalisation.
  • Figure 31 Genome tracks and peaks of MTF2 in WT-like mES cells (DMSO, green) and mESCs with an MTF2-KO (dTAG, red) using optimized epic2 parameters, i.e. FDR (q-value) of 0.01 , window size of 5,000 and gap of 1 using IgG normalisation.
  • FDR q-value
  • B HoxA gene cluster.
  • Figure 32 Number of peaks, genome tracks and peaks of MTF2 in WT-like mES cells (DMSO) and MTF2-KO mESCs (dTAG) using optimized MACS2 peak calling parameters (nonbroad option, q-value of 1e-05, IgG normalisation.
  • A Number of peaks using q-values of 1e-02 and 1e-05.
  • B Genome track at Alx1 gene.
  • C Genome track at HoxA gene cluster.
  • Figure 33 Genome tracks and peaks of H3K27me3 and SLIZ12 in WT and Suz12 _/ - mESCs using optimized MACS2 peak calling parameters (non-broad option, q-value of 1e-5, IgG normalisation).
  • A Alx1 gene.
  • B HoxA gene cluster.
  • FIG 34 Schematic overview of the protocol used for differentiation from mouse embryonic stem cells (mESCs) to mouse embryoid bodies (mEBs). Created with BioRender.com.
  • Figure 35 Principal Component Analysis of RING1B (A) and SLIZ12 (B) using CUT&Tag in mESC and mEB of WT and MLL-AF9-expressing clones.
  • Figure 36 mRNA expression of sternness markers Nanog, Oct4, and Sox2 during differentiation from mESCs (green) to mEBs (red) in WT and MLL-AF9 clones.
  • A Decrease in sternness markers in mEBs in WT cells. Bars represent counts in mean ⁇ SD of duplicates.
  • B Maintained expression of Nanog, Oct4 and Sox2 in the clones expressing MLL-AF9 compared to WT. Bars represent counts in log2(MLL-AF9 clone/WT) mean ⁇ SD of replicates from average of both MLL-AF9 clones.
  • Figure 37 mRNA expression of lineage commitment genes during differentiation from mESCs to mEBs in clones expressing MLL-AF9. Data represent the mean ⁇ SD counts expressed in log2(MLL-AF9 clone/WT) of replicates from both clones.
  • A Mesoderm lineage markers.
  • B Endoderm lineage markers.
  • C Ectoderm lineage markers.
  • Figure 38 Expressing MLL-AF9 slows down differentiation and activates different regions than in WT.
  • A Chromatin accessibility using ATAC-seq. Heatmap of chromatin accessibility measured by ATAC-seq in WT and MLL-AF9 clones B121C and D1C in mESC (day 0) and mEB (day 5). Peaks were included that show a significant difference above log2FC>1.5(P ⁇ 0.001) when comparing any of the MLL-AF9 clones in mEBs with WT mEBs, limited to top 2000 peaks by height.
  • B Gene expression using mRNA-seq.
  • DO mESC.
  • D5 EBs. It should be noted that what is displayed are not necessarily the same genes in the same order, but a list of the top peaks in each ATAC-seq and mRNA-seq.
  • Figure 39 Excerpt of figure 33B on mRNA expression in mESC and mEBs in WT and clones expressing MLL-AF9. The heatmap displays mRNA features showing a significant difference above log2FC>1.5 (P ⁇ 0.001) when comparing any of the MLL-AF9 clones with WT in mEBs.
  • FIG. 40 MLL-AF9 expression does not affect expression of Hoxa9 or Hoxa10, but of Ezh2 and Cdkn2a.
  • A Expression of Hoxa9 and Hoxa10 in WT and MLL-AF9 clones. Bars represent counts in mean ⁇ SD of duplicates.
  • B Expression of Cdkn2a and Ezh2 in the MLL- AF9 clones in relation to WT cells. Bars represent Iog2fold changes in mean ⁇ SD of duplicates.
  • FIG 41 Heatmaps of top CUT&Tag enrichments for RYBP, MLL-AF9 (FLAG), H2AK119ub, H3K27me3, H3K79me2, Mel18, RING1 B, SUZ12. Signal of two replicates per antibody are shown separately.
  • A WT and both MLL-AF9 clones at ESC stage.
  • B WT and both MLL-AF9 clones at the EB stage. The green circled region indicates some interesting changes in MLL-AF9 clones.
  • Figure 42 Changes in gene expression at MLL-AF9- (A,B) and SLIZ12- (C,D) bound sites in embryoid bodies of MLL-AF9 clones B121C (A,C) and D1C (B,D) compared to WT embryoid bodies.
  • the MLL-AF9 plots (A, B) show only positive fold changes, as MLL-AF9 is not expressed in WT cells and thus cannot be lost in the clones compared to WT.
  • Figure 43 Scatterplot showing changes in binding of PRC1 protein (A) RYBP, (B) RING1B and (C) MEL18, and of (D) H2AK119ub in combination with changes in ATAC-seq peaks and mRNA expression in clones expressing MLL- AF9 in EBs. Left: MLL- AF9 clone B121C. Right: MLL- AF9 clone D1C.
  • Figure 44 Scatterplot showing changes in binding of (A) SUZ12 and (B) H3K27me3 in combination with changes in ATAC-seq peaks and mRNA expression in clones expressing MLL-AF9 in EBs. Left: MLL- AF9 clone B121C. Right: MLL- AF9 clone D1C.
  • Figure 45 Scatterplot showing H3K79me2 changes in combination with changes in ATAC- seq peaks and mRNA expression in MLL-AF9 clones in EBs. Left: MLL- AF9 clone B121C. Right: MLL- AF9 clone D1C.
  • the term “about”, when referring to a value is meant to encompass variations of in one example ⁇ 20% or ⁇ 10%, in another example ⁇ 5%, in another example ⁇ 1 %, and in still another example ⁇ 0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods.
  • concentrations indicated as a percentage may refer to a percentage of volume per volume (% v/v).
  • 1 % formaldehyde can be understood to mean 1% (v/v) formaldehyde, e.g. 1 ml formaldehyde per 100 ml solution.
  • the term “crowding agent” as used herein may refer to an agent that is used to mimic the physiological conditions of the cells. Nuclei and cells can be filled with about 200 to 400 mg/ml of different macromolecules (chromatin, associated proteins, active transcription machinery etc.) while the volume occupied by liquid is relatively small. A crowding effect may occur when high concentrations of macromolecules are present. Therefore, in the presence of a crowding agents, in vitro results obtained with methods that profile chromatin-associated proteins for their chromatin binding sites may tend to better reflect the in vivo situation. For example, crowding agents may aid the stabilisation of membranes and proteins.
  • macromolecules chromatin, associated proteins, active transcription machinery etc.
  • Crowding agents may also stabilise the native protein conformation or protein folding by displacing liquid because unfolded proteins would need more space and are thermodynamically less favourable when crowding agents are present. Further, at least at lower salt concentrations crowding may increase protein binding to DNA and may thus prevent chromatin proteins such as PRC2 proteins from detaching from DNA. In addition, crowding agents may increase the viscosity of the solution and may thus reduce protein diffusion. Such an effect may enhance protein-protein and/or protein-DNA interactions. Exemplary crowding agents include sucrose, hexylene glycol, polyethylene glycol (PEG), dextran, Ficoll and glycerol.
  • Permeabilization may allow antibodies or transposomes that are linked to specific binding agents to pass through the cellular membrane and enter the cell. Permeabilization of the cell membranes may be achieved by a chemical treatment (e.g. by adding detergents or solvents) or a physical treatment.
  • the cell may be permeabilized with a permeabilization agent.
  • the cell may be permeabilized by electroporation or biolistics.
  • the permeabilization agent may be a lysolipid or a non-ionic detergent.
  • a cell with a permeabilized cell membrane will generally retain the cell membrane such that the cell's structure remains substantially intact.
  • a cell with a permeabilized membrane is not a "lysed" cell, for example as occurs in standard DNA purification techniques.
  • a cell membrane refers to reducing the integrity of a cell membrane such that the cell's structure does not remain intact (e.g., such as during cell lysis).
  • cells can first be treated with a permeabilization reagent and then stained with trypan blue (Merck Millipore). The cells-trypan blue mix can be transferred on a microscopy glass, covered with a glass slip, and looked at under a microscope. When the cells turn blue, the permeabilization is successful.
  • Exemplary cell permeabilization reagents include organic solvents, such as methanol and acetone, and detergents, such as saponin (e.g. digitonin), Triton X-100 and Tween-20.
  • the cell permeabilization reagent is a non-saponin detergent such as Triton X-100 and Tween-20.
  • the term “Good’s buffer” as used herein may refer to buffering agents for biochemical and biological research such as those selected and described by Norman Good and colleagues.
  • Exemplary Good’s buffers are HEPES, Tricine, MES, ADA, PIPEs, ACES, MPOSO, Cholamine chloride, MOPS, BES, TES, DIPSO, TASO, Acetamidoglycine, POPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tris, Glycinamide, Glycylglycine, Bicine and TAPS.
  • the Good’s buffer used is HEPES.
  • a Good’s buffer is used that can chelate Mg 2+ in solution, for example residuals from the cells, which may help avoiding premature activation of the transposase and unspecific cutting in accessible chromatin.
  • a Good’s buffer may further improve the tagmentation process through providing a wider pH range.
  • transposase as used herein may refer to an enzyme that catalyses the process of transposition, i.e. the movement of a transposon to a certain part of a genome typically by a cut-and-paste mechanism. Transposases are classified under EC No. 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known. An exemplary transposase is Transposase (Tnp) Tn5.
  • Tnp Transposase
  • the term “transposome” as used herein may refer to a transposase-transposon complex. A conventional way for transposon mutagenesis usually places the transposase on the plasmid.
  • transposase in some such systems, termed “transposomes", the transposase can form a functional complex with a transposon recognition site that is capable of catalysing a transposition reaction.
  • the transposase or integrase may bind to the transposase recognition site and insert the transposase recognition site into a target nucleic acid. This process is also referred to as “tagmentation”.
  • transposon may refer to a first DNA molecule comprising a first transposase recognition site and a second DNA molecule comprising a second transposase recognition site. Integration of the transposon (or the two parts of a broken transposon) yields a cleaved (or fragmented) DNA with the first and second DNA molecules integrated on either side of the fragmentation site. In this way, the chromatin DNA is both fragmented and tagged at the fragmentation site.
  • the transposase recognition sites have the same sequence, while in other examples, the transposase recognition sites have different sequences.
  • the DNA can be effectively fragmented into small fragments amenable to analysis by next generation sequencing methods.
  • the chromatin DNA is contacted with at least two different transposomes, and wherein the different transposomes comprise different DNA sequences.
  • the tagged chromatin DNA can be tagged at the 5' and 3’ end with different transposon sequences.
  • the first and second DNA molecules of the transposon can further include a variety of tag sequences, which can be added covalently to the fragments in the process of the disclosed method.
  • tags may refer to a nucleotide sequence that is attached to another nucleic acid to provide the nucleic acid with a functionality.
  • tags include barcodes, primer sites, affinity tags, and reporter moieties or any combination thereof, such as those described above.
  • tagging may refer to the step of attaching the tag to a nucleic acid to provide the nucleic acid with a functionality.
  • the term “activating the transposase” as used herein may refer to a process that results in excising and tagging the sequence of DNA bound to the chromatin-associated factor of interest wherein the transposase integrates the first and second DNA molecules into chromatin DNA. Activation may be achieved by addition of a divalent cation, e.g. Mg 2+ .
  • chromatin as used herein may refer to the complex assembly of DNA and proteins. It may serve as the architectural foundation of eukaryotic cells and allows to orchestrate essential processes such as transcription, replication, and repair. Chromatin may also contain RNA.
  • histone may refer to small, positively charged proteins around which DNA is wound to form nucleosomes. Histones are central to chromatin organization. About 150 bp of a DNA molecule is wrapped around a histone octamer consisting of two H2A/H2B dimers and two H3/H4 dimers like beads on a string. This nucleosomal arrangement facilitates both compaction and accessibility of the genetic material.
  • the DNA part between nucleosomes is called linker DNA and it accessible for nucleases unlike the DNA around the histones. It varies in length among species with an average of 33 bp.
  • the linker DNA is bound by histone H1 to stabilise the nucleosome and to further condensate the chromatin into a 30 nm fiber.
  • histone H1 By being organised as nucleosomes, the DNA molecule is compressed to a third of its length.
  • Known human histones include five classes H1/H5, H2A, H2B, H3 and H4.
  • the class H1 includes H1 F0, H1 FNT, H1 FOO, H1 FX, HIST1 H1A, HIST1 H1 B, HIST1 H1 C, HIST1 H1 D, HIST1 H1 E and HIST1 H1 T.
  • Class H2A includes H2AFB1 , H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, HIST1 H2AA, HIST1 H2AB, HIST1 H2AC, HIST1 H2AD, HIST1 H2AE, HIST1 H2AG, HIST1 H2AI, HIST1 H2AJ, HIST1 H2AK, HIST1 H2AL, HIST1 H2AM, HIST2H2AA3 and HIST2H2AC.
  • Class H2B includes H2BFM, H2BFS, H2BFWT, HIST1 H2BA, HIST1 H2BB, HIST1 H2BC, HIST1 H2BD, HIST1 H2BE, HIST1 H2BF, HIST1 H2BG, HIST1 H2BH, HIST1 H2BI, HIST1 H2BJ, HIST1 H2BK, HIST1 H2BL, HIST1 H2BM, HIST1 H2BN, HIST1 H2BO and HIST2H2BE.
  • Class H3 includes HISTH3A, HISTH3B, HISTH3C, HISTH3D, HISTH3E, HISTH3F, HISTH3G, HISTH3H, HISTH3I, HISTH3J, HIST2H3C and HIST3H3.
  • Class H4 includes HIST1 H4A, HIST1 H4B, HIST1 H4C, HIST1 H4D, HIST1 H4E, HIST1 H4F, HIST1 H4G, HIST1 H4H, HIST1 H4I, HIST1 H4J, HIST1 H4K, HIST1 H4L and HIST4H4. There are also variants of histones with special roles.
  • Variant histones include H3.3, centromeric H3 variant (cenH3, called also CENPA), H3.1, H3.2, TS H3.4, H3.5, H3.Y, H2A.X, H2A.Z, H2A.Z.2, H2A.B, H2A.L, H2A.P, H2A.1 , H2B.1, H2A.J, H2B.1 , H2B.W, H2B.Z, H2B.E. H1 and H5 linker histones are generally located between nucleosomes. Histones may control accessibility and expression of the region of DNA (or specific locus) around which they are bound or regions of DNA with which they associate.
  • histones with repressive modifications to their N-terminal tails tend to promote tightly compacted chromatin that prevents transcriptional machinery from accessing the DNA.
  • histones with modifications to their N-terminal tails that increase access to the DNA tend to promote chromatin with high levels of gene expression.
  • histone modification may refer to a post-transcriptional modification of the N-terminal tails of the histone proteins. Histone modifications may play a crucial role in regulating chromatin structure and function. Exemplary histone modifications include methylation, acetylation, propionylation, butyrylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, succinylation and ribosylation. Specific histone modifications include lysine methlyation, arginine methlyation, lysine acetylation, and serine/threonine/tyrosine phosphorylation. Exemplary specific histone modifications are trimethylation of histone H3 lysine 27 (H3K27me3) or trimethylation of histone H3 lysine 9 (H3K9me3). Histone modifications may be removed again.
  • writer may refer to enzymes responsible for catalyzing a histone modification.
  • exemplary writers include histone acetyltransferases (HATs) and histone methyltransferases (HMTs).
  • Histone methyltransferases (HMTs) may add methyl groups to lysine and arginine residues.
  • HMTs histone acetyltransferases
  • HMTs histone methyltransferases
  • DOT 1 L which catalyses the methylation of lysine 79 of histone H3 despite the absence of a SET domain, causing the histone marks H3K79me1/2/3.
  • Enhancer of Zeste Homologue 1 (EZH1) and its homologue EZH2 are the only histone methylases responsible for the mono-, di- and trimethylation of lysine 27 of histone H3, associated with gene repression and chromatin compaction.
  • EZH1 and EZH2 are part of the Polycomb Repressive Complex 2.
  • the enzymes responsible for catalysing acetylation of lysine residues are called histone acetyltransferases (HATs).
  • HAT enzymes include but are not limited to the GNAT superfamily including Gcn5, SAGA, SLIK, STAGA, ADA, A2, Gcn5L, p300/CREB-binding protein associated factor (PCAF), Elp3, HPA2, and HAT1), the MYST family including MOZ (Monocytic Leukemia Zinc Finger Protein), Ybf2/Sas3, Sas2, Tip60, Esa1 , MOF, MORF, and HBO1 , the p300/CBP family including adenoviral E1A associated protein of 300 kDa (p300) and the CREB-binding protein (CBP), and other HATs such as steroid receptor coactivator 1 (SRC 1), ATF-2, and TAFII250.
  • GNAT superfamily including Gcn5, SAGA, SLIK, STAGA, ADA, A2, Gcn5L, p300/CREB-binding protein associated factor (PCAF), Elp3, HPA2, and HAT
  • Acetyl-Coenzyme A is a common source of the acetyl group transferred to a target histone lysine residue by HAT enzymes. Acetylation removes the positive charge of lysines. This way, the interaction of the histone proteins with the DNA is weakened since the backbone of DNA consists of negatively charged phosphate groups. It causes a more open chromatin, called euchromatin, and a higher accessibility for the transcription machinery. Generally, euchromatin and acetylated histone tails are associated with actively transcribed genes. Histone ubiquitintransferases may catalyse monoubiquitinylation. Phosphate groups may be added to serine residues by histone kinases.
  • erasers may refer to enzymes responsible for removing a histone modification.
  • exemplary erasers include histone deacetylases (HDACs) and histone demethylases (HDMs).
  • Histone monoubiquitinylation may be removed by deubiquitinating enzymes.
  • Phosphate groups may be removed by phosphatases.
  • reader or “reader protein” as used herein may refer to a protein that is able to recognize a histone modification by binding specifically to it. Readers may recognise modified histone tails by certain domains. When a protein consists of a bromodomain, it may bind to acetylated lysines. Thereafter, it may recruit other proteins and cause other actions. Methylated histones may be bound by proteins with a chromodomain. Some chromodomains are specific for certain histone marks, such as the one of CBX proteins in canonical Polycomb Repressive Complex 1 that only recognises H3K27me3.
  • transcription factor may refer to a protein that regulates transcription.
  • a transcription factor may be a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.
  • transcription factors may regulate the binding of RNA polymerase and the initiation of transcription.
  • a transcription factor may bind upstream or downstream to either enhance or repress transcription of a gene by assisting or blocking RNA polymerase binding.
  • transcription factor includes both inactive and activated transcription factors.
  • a transcription factor may affect regulation of gene expression.
  • Exemplary transcription factors include but are not limited to AAF, abl, ADA2, ADA-NF1 , AF-1 , AFP1 , AhR, ATIN3, ALL-1, alpha-CBF, alpha-CP 1, alpha-CP2a, alpha-CP2b, alphaHo, alphaH2-alphaH3, Alx-4, aMEF-2, AML1, AMLIa, AMLIb, AMLIc, AMLIDeltaN, AML2, AML3, AML3a, AML3b, AMY-1 L, A-Myb, ANF, AP-1, AP-2alphaA, AP-2alphaB, AP-2beta, AP-2gamma, AP-3 (1), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, Arnt, Arnt (774 M form), ARP-1, ATBF1-A, ATBF1-B, ATF, ATF-1 , ATF-2, ATF-3, ATF-3deltaZIP
  • ENKTF-1 EPASI, epsilonFI, ER, Erg-1, Erg-2, ERR1, ERR2, ETF, Ets-1, Ets-1 deltaVil, Ets-2, Evx-1 , F2F, factor 2, Factor name, FBP, f-EBP, FKBP59, FKHL18, FKHRL1P2, Fli-1 , Fos, FOXB1, FOXC1 , FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3, FOXF1, FOXF2, FOXGla, FOXGlb, FOXGlc, FOXHI1 , FOXI1 , FOXJIa, FOXJIb, FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXKIa, FOXKIb, FOXKIIc, FOXL1, FOXMlla, FOXMlIb, FOXMl
  • chromatin-associated factor may refer to any molecule, for instance a protein, that is able to bind one or more chromatin binding sites.
  • exemplary chromatin-associated factors include histones and non-histone proteins.
  • Exemplary non-histone proteins include transcription factors, writers, readers and erasers such as those mentioned above.
  • ChIP Chromatin immunoprecipitation
  • Chromatation may refer to a method involving crosslinking of chromatin that is used to determine whether a particular protein binds to or is localized to a specific DNA sequence. ChIP has been used to study DNA-protein interactions for decades. In a conventional ChIP workflow, harvested cells are crosslinked with formaldehyde to ensure covalent reversible proteins crosslinking to DNA. Cells are lysed and the chromatin is sheared by sonication. These steps are known as chromatin preparation. Antibodies against a protein or histone modification of interest bind to their epitope on the chromatin. The antibodies also bind to the Protein A-coated magnetic beads.
  • Immunoprecipitated DNA is typically eluted from the beads using SDS- containing buffer, the crosslinking is typically released by heating at 65°C in the presence of SDS and sodium chloride.
  • the recovered DNA can be purified and can be used for the enrichment check by qPCR, library preparation and sequencing.
  • CUT&Tag The term "Cleavage Under Targets and Tagmentation” or "CUT&Tag”, as used herein, may be used to describe methods for chromatin profiling without the need for chromatin preparation.
  • cells are bound to magnetic concanavalin A-coated beads during the process up to DNA extraction and purification.
  • An antibody against the desired chromatin mark enters the permeabilized cells and marks the site in the genome.
  • a subsequently added fusion protein consisting of protein A and the transposase Tn5 is bound to the antibody (e.g. via a second antibody). After binding of the protein A part to the antibody (e.g. via the second antibody) and activation of Tn5, the latter cuts the DNA and incorporates sequencing adapters at the site.
  • CUT&Tag can be used to study transcription factors and chromatin alterations in various species, including humans and mice. Permeabilization in conventional CUT&Tag protocols can be performed using digitonin. In some embodiments, CUT&Tag can be performed using a non-saponin detergent, for instance Triton X-100 or Tween-20.
  • Chromatin immunocleavage may refer to a chromatin profiling technique. It may use a fusion protein of micrococcal nuclease and protein A (pAMNase) to fragment the chromatin at target sites. After a permeabilisation of the cells, an antibody enters the cells, binds to its epitope on the chromatin mark of interest, and thereby marks the site in the genome.
  • the fusion protein pA-MNase can be tethered to the antibody and bind it. After activation, the MNase part cuts the DNA and DNA sequences are released into solution.
  • MNase preferably binds at linker regions; therefore, peaks appear ca. 100 to 200 bp from known binding sites.
  • the resolution in ChIP may be in range of 1,000 bp.
  • CUT&RUN chromatin profiling technique
  • cells are bound to magnetic beads coated with concanavalin A throughout the process until DNA extraction and purification, allowing wash steps without centrifugation and the sample loss associated with centrifugation.
  • incubation conditions typically need to be adjusted during the MNase reaction. This may require precise, temperature-sensitive work and depend on the abundance and availability of the protein of interest. Subsequent library preparation is typically performed prior to analyzing samples using sequencing technologies, which may increase time, cost, labor and potential sample loss.
  • binding site may refer to a region on a protein, DNA, or RNA to which other molecules specifically bind.
  • the binding site a chromatin-associated factor of interest binds to may be referred to as chromatin binding site.
  • Methods for determining the chromatin binding sites of a chromatin-associated factor of interest known in the art include ChIP, ChIC, CUT&RUN, and conventional CUT&Tag.
  • crosslinking may refer to a method of chemically joining two or more molecules by a covalent bond. Crosslinking methods may allow for the stabilization of molecular structures and the capture of molecular interactions within a cell so that they can be preserved and studied. DNA-protein crosslinking can be caused by a variety of chemical and physical agents. Crosslinking agents or reagents appropriate for use with the disclosed methods include but are not limited to formaldehyde, paraformaldehyde, methylene blue with or without exposure to white light, sodium fluorescein, acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, ultraviolet light, and lasers.
  • strong crosslinking conditions may refer to crosslinking conditions that may result in masking antibody epitopes, making chromatin less accessible for antibodies and/or the transposome that is linked to a specific binding agent. Strong crosslinking conditions may include the use of 1% formaldehyde. For example, strong crosslinking conditions may include the use of 1% (v/v) formaldehyde. Incubation times for strong crosslinking conditions may be around 8 minutes for histone modifications and around 15 minutes for non-histone proteins.
  • light crosslinking conditions or “mild crosslinking conditions” as used herein may refer to crosslinking conditions that may lower the risk of masking antibody epitopes and thus making chromatin less accessible for antibodies and/or the transposome that is linked to a specific binding agent.
  • mild crosslinking conditions may include the use of less than 0.5% formaldehyde or 0.4% formaldehyde or less or 0.3% formaldehyde or less or 0.2% formaldehyde or less, e.g. 0.1% formaldehyde or less.
  • mild crosslinking conditions may include the use of less than 0.5% (v/v) formaldehyde or 0.4% (v/v) formaldehyde or less or 0.3% (v/v) formaldehyde or less or 0.2% (v/v) formaldehyde or less, e.g. 0.1% (v/v) formaldehyde or less.
  • Incubation times for mild crosslinking conditions may be ⁇ 8 minutes for crosslinking of histones or ⁇ 15 minutes for crosslinking of non-histone proteins, including all whole and non-integer numbers greater than zero.
  • exemplary incubation times for mild crosslinking conditions may be ⁇ 5 min for crosslinking of histones or ⁇ 10 min or ⁇ 5 min for crosslinking of non-histone proteins.
  • subjecting cells to mild crosslinking conditions may involve using 0.1%-0.4% (v/v) formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min for crosslinking of non-histone proteins.
  • FFA formaldehyde
  • CH 2 O a chemical having the formula CH 2 O. It can be used as a chemical crosslinking reagent to crosslink DNA to protein or DNA to DNA within a chromatin complex to maintain chromatin structure.
  • PBS Phosphate-Buffered Saline-PBS is a buffer solution which is typically used in biological research to simulate physiological conditions.
  • telomere binding agent may refer to an agent that binds substantially or preferentially only to a defined target such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule.
  • a nucleic acid-specific binding agent binds substantially only to the defined nucleic acid, such as DNA, or to a specific region within the nucleic acid.
  • a specific binding agent is a probe or primer that specifically binds to a target nucleic acid of interest.
  • a specific binding agent is a transcription factor that specifically binds to a target nucleic acid of interest, such as chromatin DNA.
  • a protein-specific binding agent binds substantially only the defined protein, or to a specific region within the protein.
  • a "specific binding agent” includes antibodies and other agents that bind substantially to a specified polypeptide.
  • Antibodies can be monoclonal or polyclonal antibodies that are specific for the polypeptide, as well as immunologically effective portions ("fragments") thereof.
  • fragments immunologically effective portions
  • the determination that a particular agent binds substantially only to a specific polypeptide may readily be made by using or adapting routine procedures.
  • One suitable in vitro assay makes use of the Western blotting procedure (as described in many standard texts).
  • a specific binding agent may include all or parts of protein A (pA) or protein G (pG) or derivatives thereof which have affinity for antibodies.
  • Protein A is a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArIR. It is commonly used in biochemical research because of its ability to bind immunoglobulins. Like Protein A but with differing binding specificities, Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria. Protein G is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein commonly used for purifying antibodies through its binding to the Fab and Fc region.
  • antibody refers to a polyclonal, monoclonal, recombinant, or synthetic immunoglobulin molecule that specifically binds a target antigen.
  • the term includes intact immunoglobulin molecules, fragments or polymers of those immunoglobulin molecules, chimeric antibodies containing sequences from more than one species, class, or subclass of immunoglobulin, and human or humanized versions of immunoglobulin molecules or fragments thereof containing a least the idiotype of an immunoglobulin that specifically binds the target antigen.
  • the agents binding to chromatin in particular the antibody or chemical substance, used in the methods described herein may specifically bind to histones, modified histones and/or other factors, in particular polypeptides such as enzymes, interacting with such histones and/or modified histones.
  • adapter may refer to a short double-stranded DNA oligonucleotide whose sequence is known.
  • sequencing library may refer to a nucleic acid representation, wherein each nucleic acid is identifiable by, e.g., the use of an individual sequence tag. Accordingly, obtaining a sequencing library typically requires a process capable of ensuring that specific adaptor sequences are added to the ends of the nucleic acid fragments to be analyzed. Most of the next generation sequencing applications require the preparation of a sequencing library comprising nucleic acids with specific adapters at 5' and 3' ends.
  • the Illumina sequencing workflow utilizes partially complementary adaptor oligonucleotides that are used for priming the PCR amplification and introducing the specific nucleotide sequences required for cluster generation by bridge PCR and facilitating the sequencing-by-synthesis reactions. Accordingly, the resulting sequencing library is suitable for use in standard sequencing applications, e.g. next generation sequencing.
  • normalization may refer to a process that results in removal of at least some biases in the data such as unspecific chromatin binding sites of the chromatin- associated factor of interest, while specific binding sites persist.
  • the term “replicate” may be used for samples of a cell where the biological material is the same. For example, the steps used to determine at least one chromatin binding site of a chromatin-associated factor of interest in a cell are repeated with at least one, at least two replicates, at least three replicates or at least four replicates. Alternatively or in addition, the method for determining at least one chromatin binding site of a chromatin- associated factor of interest in a cell as described herein is performed with a first replicate and a normalization control is generated with a second replicate.
  • first replicate may be used for a first sample of a cell, such as DNA of a permeabilized cell
  • second replicate or “third replicate” or “fourth replicate”
  • second sample or third sample or fourth sample, respectively
  • the method is conducted twice (or three or four times, respectively).
  • the term “false positive” may denote a result of the methods for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell as described herein which incorrectly indicates a chromatin binding site of the chromatin- associated factor of interest.
  • the term “input normalization” or “normalization” may refer to a method in which the chromatin binding sites are determined in a cell which has not been contacted with a first antibody or second antibody in a method as described herein, and the determined chromatin binding sites are used for the purpose of normalization in a method as described herein.
  • the term “input normalization” or “normalization” may refer to a method in which the chromatin binding sites are determined in DNA extracted and/or purified from a cell which has not been contacted with a first antibody or second antibody in a method as described herein, and the determined chromatin binding sites are used for the purpose of normalization in a method as described herein.
  • an input sample can be subjected to method steps (c) to (f) or method steps (d) to (f) of a method as described herein in order to allow for a normalization of the at least one chromatin binding site of the chromatin-associated factor of interest in the cell determined in step f).
  • normalization may be carried out by performing in a first replicate method steps (a) to (f) of a method as described herein and the following modified method steps in a second replicate: in step (a), instead of permeabilizing of the cell, purifying DNA from the cell, omitting step (b) of the method as described herein, i.e. omitting the step of contacting the permeabilized cell with a first antibody that specifically binds the chromatin- associated factor of interest and with a second antibody that specifically binds to the first antibody, performing step (c) of the method as described herein with the purified DNA, i.e.
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; performing step (d) of the method as described herein, i.e.
  • the transposase activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA in the second replicate; wherein the sequence of the excised and tagged DNA segment determined in the second replicate is used as a normalization control in the step of determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell determined in step (f) in the first replicate.
  • normalization may be carried out by performing in a first replicate method steps (a) to (f) of a method as described herein and the following modified method steps in a second replicate: purifying DNA from the cell, contacting the purified DNA with the transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA in the second replicate; wherein the sequence of the excised and tagged DNA segment determined in the second replicate is used as a normalization control in the step of determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell determined in step (f) in the first replicate.
  • the term “input sample” may refer to sequence information or a sample used to provide sequence information wherein the sequence information can be used as a normalization control in the step of determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell determined in step f) of the method as described herein.
  • An exemplary method for generating a normalization control suitable for use in the above described method for determining at least one chromatin binding site of the chromatin- associated factor of interest in the cell comprises the following steps: purifying DNA from the cell; contacting the purified DNA with a transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA.
  • Another exemplary method for generating a normalization control suitable for use in the above described method for determining at least one chromatin binding site of the chromatin- associated factor of interest in the cell comprises the following steps: purifying DNA from the cell;
  • transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA.
  • Another exemplary method for generating a normalization control suitable for use in the above described method for determining at least one chromatin binding site of the chromatin- associated factor of interest in the cell comprises the following steps: purifying DNA from the cell; contacting the purified DNA with a transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA; wherein the steps of contacting the purified DNA with the transposome and activating the transposase are performed in presence of a crowding agent.
  • Another exemplary method for generating a normalization control suitable for use in the above described method for determining at least one chromatin binding site of the chromatin- associated factor of interest in the cell comprises the following steps: purifying DNA from the cell;
  • the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; if a DNA segment is excised and tagged with the first and second DNA molecule by activating the transposase, determining the sequence of the excised and tagged DNA; wherein the steps of contacting the purified DNA with the transposome and activating the transposase are performed in presence of a crowding agent.
  • IgG normalization may refer to a method in which the chromatin binding sites are determined in a cell which has been subjected to a first antibody that does not target a chromatin-associated factor of interest but instead constitutes an IgG antibody that is not supposed to target any specific molecule in the cell in a method as described herein, and the determined chromatin binding sites are used for the purpose of normalization in a method as described herein.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in the presence of a crowding agent.
  • the steps of contacting the purified DNA with the transposome and activating the transposase are performed in the presence of a crowding agent.
  • the crowding agent is selected from a group comprising sucrose, hexylene glycol and glycerol. In some embodiments, the crowding agent is sucrose.
  • a crowding agent in the steps of contacting a permeabilised cell with a transposome and activating the transposase as described herein may mimic the “crowding” of cytoplasm and nucleus with macromolecules.
  • the use of a crowding agent may thus mimic a situation closer to the actual in vivo situation.
  • a crowding agent may stabilize DNA-protein and/or protein-protein interactions.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in the presence of a Good’s buffer.
  • the steps of contacting the purified DNA with the transposome and activating the transposase are performed in the presence of a Good’s buffer.
  • the Good’s buffer is a buffer that can chelate Mg 2+
  • the Good’s buffer is a buffer other than HEPES.
  • the Good’s buffer is selected from Tricine, MES, ADA, PIPEs, ACES, MPOSO, Cholamine chloride, MOPS, BES, TES, DIPSO, TASO, Acetamidoglycine, POPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tris, Glycinamide, Glycylglycine, Bicine and TAPS.
  • the Good’s buffer is Tricine.
  • a Good’s buffer as described herein may help avoid premature activation of the transposase and unspecific cutting in accessible chromatin. The presence of Good’s buffer may be beneficial for the tagmentation process through a wider pH range.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in the presence of a crowding agent and a Good’s buffer. In some embodiments, the steps of contacting the purified DNA with the transposome and activating the transposase are performed in the presence of a crowding agent and a Good’s buffer.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in the presence of sucrose and tricine. In some embodiments, the steps of contacting the purified DNA with the transposome and activating the transposase are performed in the presence of sucrose and tricine.
  • a cell comprises a plurality of cells.
  • a plurality of cells is a mixed population of cells.
  • a mixed population of cells as used herein comprises cells from different cell types.
  • a cell used in a method as described herein may be obtained from a biological sample obtained directly from a subject.
  • biological samples are samples of: blood, saliva, lymph, cerebrospinal fluid, vitreous humor, aqueous humor, mucous, tissue, etc.
  • a cell is a primary immune cell, such as but not limited to a T-cell.
  • a method as described herein is performed on invertebrate cells, including but not limited to Drosophila cells.
  • a method as described herein is performed on vertebrate cells.
  • a method as described herein is carried out on mammalian cells, including but not limited to cells from cell lines, primary immune cells (e.g., T-cells), stem cells, diseased cells, healthy cells, etc.
  • a method as described herein is performed on mixed human cells and cells of another organism, examples of which are non-human primate cells, mouse cells, etc.
  • a method as described herein is performed on human cells.
  • mammalian cells that may be used in methods as described herein are cells obtained directly from a subject, cells obtained from a mammalian cell line, cultured mammalian cells, transgenic mammalian cells, etc.
  • Cells used in certain methods as described herein may be obtained from a living animal, e.g., a mammal, or may be obtained from a collection of isolated cells.
  • An isolated cell may be a primary cell, such as those recently isolated from an animal (e.g., cells that have undergone none or only a few population doublings and/or passages following isolation), or may be cells of a cell line that is capable of prolonged proliferation in culture (e.g., for longer than 3 months) or indefinite proliferation in culture (immortalized cells).
  • a cell is a somatic cell.
  • Somatic cells may be obtained from an individual, e.g., a human, and cultured according to standard cell culture protocols known to those of ordinary skill in the art.
  • Cells may be obtained from surgical specimens, tissue or cell biopsies, etc, Cells may be obtained from any organ or tissue of interest, including but not limited to: skin, lung, cartilage, brain, CNS, PNS, breast, blood, blood vessel (e.g., artery or vein), fat, pancreas, liver, muscle, gastrointestinal tract, heart, bladder, kidney, urethra, and prostate gland.
  • organ or tissue of interest including but not limited to: skin, lung, cartilage, brain, CNS, PNS, breast, blood, blood vessel (e.g., artery or vein), fat, pancreas, liver, muscle, gastrointestinal tract, heart, bladder, kidney, urethra, and prostate gland.
  • a cell used in conjunction with the methods as as described herein is a healthy normal cell, which is not known to have a disease, disorder, or abnormal condition.
  • a cell used in conjunction with methods as described herein is an abnormal cell, for example, a cell obtained from a subject diagnosed as having a disorder, disease, or condition, including, but not limited to a degenerative cell, a neurological diseasebearing cell, a cell model of a disease or condition, an injured cell, etc.
  • a cell is an abnormal cell obtained from cell culture, a cell line known to include a disorder, disease, or condition.
  • a cell is a control cell.
  • a cell can be a model cell for a disease or condition.
  • Examples of a cell that may be used in an embodiment of a method as described herein are one or more of: eukaryotic cells, vertebrate cells, which in some embodiments may be mammalian cells.
  • a non-limiting example of cells that may be used in methods as described herein are: vertebrate cells, invertebrate cells, and non-human primate cells.
  • Additional, examples of cells that may be used in an embodiment of a method as described herein are one or more of: rodent cells, dog cells, cat cells, avian cells, fish cells, cells obtained from a wild animal, cells obtained from a domesticated animal, and other suitable cell of interest.
  • a cell is a human cell.
  • a cell is a stein cell, an embryonic stem cell, or embryonic stem cell-like cell.
  • a cell is a naturally occurring cell and, in some embodiments, a cell is an engineered cell.
  • Cells useful in embodiments of methods as described herein may be maintained in cell culture following their isolation.
  • Cells may be genetically modified or not genetically modified in various embodiments.
  • Cells may be obtained from normal or diseased tissue.
  • cells are obtained from a donor, and their state or type is modified ex vivo using a method as described herein.
  • a cell may be a free cell in culture, a free cell obtained from a subject, a cell obtained in a solid biopsy from a subject, organ, or solid culture, etc.
  • the number of cells is ⁇ 500,000. In some embodiments, the number of cells is ⁇ 100,000. In some embodiments, the number of cells is ⁇ 50,000. In some embodiments, the number of cells is ⁇ 10,000. In some embodiments, the number of cells is ⁇ 5,000. In some embodiments, the number of cells is ⁇ 1,000.
  • the cells and/or nuclei may be subjected to crosslinking. In other embodiments, the cells and/or nuclei are not subjected to crosslinking. In some embodiments, the cells and/or nuclei are subjected to crosslinking directly after harvest. In some embodiments, crosslinking is followed by cryopreservation. In some embodiments, crosslinking is performed prior to permeabilization. In some embodiments, mild crosslinking is performed prior to the permeabilization step. In some embodiments, crosslinking is followed by one or more washing steps and/or a quenching step prior to permeabilization.
  • crosslinking using mild cross-linking conditions is performed directly after harvest, optionally followed by cryopreservation and followed by permeabilization.
  • mild crosslinking conditions may include the use of less than 0.5% (v/v) formaldehyde or 0.4% (v/v) formaldehyde or less or 0.3% (v/v) formaldehyde or less or 0.2% (v/v) formaldehyde or less, e.g. 0.1% (v/v) formaldehyde or less.
  • Incubation times for mild crosslinking conditions may be ⁇ 8 minutes for crosslinking of histones or ⁇ 15 minutes for crosslinking of non-histone proteins, including all whole and non-integer numbers greater than zero.
  • mild crosslinking conditions may be ⁇ 5 min for crosslinking of histones or ⁇ 10 min or ⁇ 5 min for crosslinking of non-histone proteins.
  • subjecting cells to mild crosslinking conditions may involve using 0.1%-0.4% (v/v) formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min for crosslinking of non-histone proteins.
  • mild crosslinking conditions are 0.1% formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min.
  • mild crosslinking conditions are 0.1% (v/v) formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min.
  • the quenching agent is glycine. In some embodiments, quenching is performed for ⁇ 10 min or ⁇ 5 min. In some embodiments, mild crosslinking conditions are 0.1% formaldehyde for 5 min and are followed by quenching with glycine for additional 5 minutes. In some embodiments, mild crosslinking conditions are 0.1% (v/v) formaldehyde for 5 min and are followed by quenching with glycine for additional 5 minutes. In some embodiments, cells and/or nuclei subjected to crosslinking and quenching may be subjected to cryopreservation prior to further processing.
  • the chromatin-associated factor of interest is a protein. In some embodiments, the chromatin-associated factor of interest is a histone protein. In some embodiments, the chromatin-associated factor of interest is a non-histone protein. In some embodiments, the chromatin-associated factor of interest is a transcription factor. In some embodiments, the chromatin-associated factor of interest is a histone reader protein. In some embodiments, the chromatin-associated factor of interest is a writer protein. In some embodiments, the chromatin-associated factor of interest is an eraser protein.
  • the first antibody specifically binds the chromatin- associated factor of interest. In some embodiments, the first antibody binds to proteins. In some embodiments, the first antibody binds to histone proteins. In some embodiments, the first antibody may specifically bind to histones, modified histones and/or other factors, in particular polypeptides such as enzymes, interacting with such histones and/or modified histones.
  • the first antibody binds to modified histones. In some embodiments, the first antibody binds to H3K4me1/2/3, H2BK5me1, H3K27me1/2/3, H3K9me1/2/3, H4K20me1 , H3K79me1 , H3K36me3, H2AK5ac, H2AK9ac, H2BK5ac, H2BK12ac, H2BK20ac, H2BK120ac, H3K4ac, H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K36ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac, H4K91 ac, H2Aub or H2Bub.
  • the first antibody binds to non-histone proteins. In some embodiments, the first antibody binds to transcription factors. In some embodiments, the first antibody binds to AAF, abl, ADA2, ADA-NF1 , AF-1 , AFP1 , AhR, AIIN3, ALL-1 , alpha-CBF, alpha-CP 1 , alpha-CP2a, alpha-CP2b, alphaHo, alphaH2-alphaH3, Alx-4, aMEF-2, AML1 , AMLIa, AMLIb, AMLIc, AMLIDeltaN, AML2, AML3, AML3a, AML3b, AMY- 1 L, A-Myb, ANF, AP- 1 , AP-2alphaA, AP-2alphaB, AP-2beta, AP-2gamma, AP-3 (1 ), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, Arnt,
  • ENKTF-1 EPASI, epsilonFI, ER, Erg-1 , Erg-2, ERR1 , ERR2, ETF, Ets-1 , Ets-1 deltaVil, Ets-2, Evx-1 , F2F, factor 2, Factor name, FBP, f-EBP, FKBP59, FKHL18, FKHRL1 P2, Fli-1 , Fos, FOXB1 , FOXC1 , FOXC2, FOXD1 , FOXD2, FOXD3, FOXD4, FOXE1 , FOXE3, FOXF1 , FOXF2, FOXGla, FOXGlb, FOXGlc, FOXH1 , FOXI1 , FOXJIa, FOXJIb, FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXKIa, FOXKIb, FOXKIc, FOXL1 , FOXM
  • the first antibody binds to a transcription factor known to be associated with diseases, e.g. cancer.
  • diseases e.g. cancer
  • the methods as described herein may be used to study the interaction between DNA and transcription factors in a diseased cell and/or cells derived from diseased tissue.
  • the cells are immobilized on a solid surface, for example a bead or the wall of a microtiter plate. Methods of coupling cells to such solid surfaces are known in the art, for example in the context of high throughput techniques.
  • the bead is a magnetic bead.
  • the bead is a magnetic Concanavalin A (ConA) bead.
  • ConA Concanavalin A
  • the cells prior to the permeabilization step, the cells are bound to beads, optionally magnetic beads.
  • one or more method steps are performed in single 1.5 ml tubes. In some embodiments, one or more method steps are performed in tube strips having a volume of 0.5 ml or less such as 0.2 ml. In some embodiments, the use of 0.2 ml tube strips instead of single 1.5 ml tubes allows the use of a multichannel pipette for wash and other resuspension steps, and/or the addition of reagents, provided that the volume is high enough for the use of a multichannel pipette. A multichannel pipette may allow to deliver liquid to multiple tubes at once. This may increase the efficiency of the process.
  • the use of a multichannel pipette can reduce the number of pipetting steps and may ensure a consistent volume delivery to multiple tubes. This may reduce pipetting errors, which in turn increases the reproducibility between replicates.
  • the use of 0.2 ml tube strips and/ or the lowering of the total volume may decrease the exposure time of the samples to the buffer, reduce the detaching rate and increase the recovery.
  • one or more method steps are performed using a total buffer volume of ⁇ 1000 pl. In some embodiments, one or more method steps are preformed using a total buffer volume of ⁇ 800 pl. In some embodiments, one or more method steps are preformed using a total buffer volume of ⁇ 500 pl. In some embodiments, one or more method steps are preformed using a total buffer volume of ⁇ 200 pl. In some embodiments, one or more method steps are preformed using a total buffer volume of ⁇ 100 pl. In some embodiments, one or more method steps are preformed using a total buffer volume of ⁇ 50 pl.
  • a transposome is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule.
  • the transposase is Tn5.
  • a modified transposase is used, which has a higher activity than the naturally occurring Tn5 transposase.
  • the transposase is loaded with a first and second DNA molecule, e.g. oligonucleotides, which are inserted into the target nucleic acid, in particular the target DNA.
  • the transposome comprises a transposase and a first and second DNA molecule.
  • the first DNA molecule may comprise a first transposase recognition site and the second DNA molecule may comprise a second transposase recognition site.
  • a hyperactive Tn5 transposase and a Tn5- type transposase recognition or MuA transposase and a Mu transposase recognition site comprising Rl and R2 end sequences are used. More examples of transposition systems that can be used in the methods as described herein include Staphylococcus aureus Tn552 (Colegio et al, J. Bacteriol, 183: 2384-8, 2001 ; Kirby C et al, Mol.
  • More examples include IS5, TnlO, Tn903, IS91 1 , and engineered versions of transposase family enzymes (Zhang et al, (2009) PLoS Genet. 5:el000689. Epub 2009 Oct 16; Wilson C. et al (2007) J. Microbiol. Methods 71 :332-5).
  • the amount of transposase that is linked to a specific binding agent is ⁇ 1: 50 of the reaction volume. In some embodiments, the amount of transposase that is linked to a specific binding agent is ⁇ 1:100 of the reaction volume. In some embodiments, the amount of transposase that is linked to a specific binding agent is ⁇ 1:150 of the reaction volume. In some embodiments, the amount of transposase that is linked to a specific binding agent is ⁇ 1:200 of the reaction volume. In some embodiments, the amount of transposase that is linked to a specific binding agent is ⁇ 1 :250 of the reaction volume.
  • the dilution of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 1 : 50. In some embodiments, the dilution of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 1:100. In some embodiments, the dilution of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 1 :150. In some embodiments, the dilution of transposase that is linked to a specific binding in the reaction volume agent is ⁇ 1 :200. In some embodiments, the dilution of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 1:250.
  • the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 74 ng/pl. In some embodiments, the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 37 ng/pl. In some embodiments, the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 25 ng/pl. In some embodiments, the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 18 ng/pl. In some embodiments, the concentration of transposase that is linked to a specific binding agent in the reaction volume is ⁇ 15 ng/pl.
  • the optimal amount of transposase that is linked to a specific binding agent may vary batch-by-batch.
  • Methods to determine the optimal amount of transposase that is linked to a specific binding agent are well known in the art and the optimal amount of transposase can be determined by routine optimization. These include, for example, subjecting different dilutions of the amount of transposase that is linked to a specific binding agent to conventional CUT&Tag or a method disclosed herein, followed by the evaluation of the library concentration and the data obtained after sequencing, e.g. by visual inspection and bioinformatic analysis.
  • Cell membranes can be permeabilized or disrupted in any way known in the art.
  • the methods of permeabilizing or disrupting the cell membrane do not disrupt the structure of the genomic DNA of the cell such that nucleosomal or chromatin structure is destroyed.
  • the cell membrane may be contacted with a reagent that permeabilizes the cell membrane.
  • Lysolipids are an exemplary class of reagents that permeabilize cell membranes.
  • Exemplary lysolipids include lysophosphatidylcholine (also known in the art as lysolecithin) or monopalmitoylphosphatidylcholine.
  • lysolipids are also described in, e.g., WO 2003/052095. The precise concentration of the agent will depend on the agent used as well as the cell to be permeabilized.
  • lysolecithin As an example, 0.25, 0.5%, 0.75 or 1 % (or a concentration between 0.25% and 1%) of lysolecithin (w/v) may be used.
  • electroporation or biolistic methods can be used to permeabilize a cell membrane such that a DNA cleaving agent is introduced into the cell and can thus contact the genomic DNA.
  • electroporation methods include those described in WO 2000/062855.
  • Biolistic methods include those described in US Patent 5,179,022.
  • Non-ionic detergents are an exemplary class of reagents that disrupt cell membranes. Exemplary nonionic detergents include NP40, Tween 20 and Triton X-100. The precise concentration of the reagent will depend on the non-ionic detergent used and/or the cell to be permeabilized.
  • permeabilization reagents include digitonin or related saponin compounds.
  • cells are permeabilized using digitonin at a concentration of 0.1%.
  • Triton X-100 may be used to permeabilize cells.
  • cells are permeabilized using Triton X-100 at a concentration of 0.1% to 0.4%.
  • cells are permeabilized using 0.1% Triton X-100.
  • DNA can be purified from the cell by various techniques known in the art (see e.g. Gupta N. DNA Extraction and Polymerase Chain Reaction. J Cytol. 2019 Apr-Jun;36(2):116-117; Preetha J Shetty, The Evolution of DNA Extraction Methods. 2020 - 8(1).
  • Conventional purification methods may include the following steps: leukocyte isolation or red cell lysis, nuclear lysis, deproteinization, RNAse-A treatment, and DNA precipitation.
  • RNAse-A treatment Several commercial products are available for rapid purification of genomic DNA from blood spots, whole blood, and other sources. Exemplary methods for DNA purification from cells are also described below in the context of extracting excised and tagged DNA segments.
  • the tagmentation reaction may be stopped by various techniques known in the art.
  • Tagmentation may be quenched using a buffer containing EDTA, and DNA fragments may be released into solution by heated digestion in a SDS buffer.
  • SDS may subsequently be neutralized using a nonionic detergent (i.e. Triton-X 100).
  • Stopping the tagmentation reaction may involve adding EDTA, SDS and/or proteinase K and may be following protein digestion at higher temperatures, e.g. 55°C.
  • tagmentation is stopped by adding 0.5 M EDTA, 10% SDS and proteinase K followed by protein digestion at 55°C.
  • the excised and tagged DNA segment may be extracted. This may be achieved by various techniques known in the art.
  • the excised and tagged DNA can be purified using column purification, phenol-chloroform extraction followed by ethanol precipitation, Solid Phase Reversible Immobilisation and Chelex® 100 and other techniques known in the art.
  • Column purification relies on binding of nucleic acids, in particular DNA, (adsorption) to the solid phase (silica or other) depending on the pH and the salt content of the used buffer.
  • aqueous phase containing nucleic acid in particular DNA
  • chloroform removing phenol residues from solution.
  • phenol-chloroform extraction is followed by ethanol or isopropanol precipitation. Since DNA is insoluble in these alcohols, it will aggregate, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing ionic strength, usually by adding sodium acetate.
  • Chelex® 100 is a chelating material distributed by Bio-Rad, which is used to purify other compounds via ion exchange. It can also be used to purify DNA.
  • SPRI Solid Phase Reversible Immobilisation
  • beads are paramagnetic (magnetic only in a magnetic field).
  • Each bead is made of polystyrene surrounded by a layer of magnetite, which is coated with carboxyl molecules. It is these that reversibly bind DNA in the presence of polyethylene glycol (PEG) and salt (commonly 20% PEG, 2.5M NaCI).
  • PEG causes the negatively-charged DNA to bind with the carboxyl groups on the bead surface.
  • the immobilization is dependent on the concentration of PEG and salt in the reaction, the volumetric ratio of beads to DNA is critical. DNA purification is often supported by removal of RNA and protein by the addition of RNase and Proteinase to the solution.
  • extraction of the excised and tagged DNA segment may be performed using phenol-chloroform extraction with subsequent ethanol precipitation.
  • extraction of the excised and tagged DNA segment may be performed using the MicroChIP DiaPure columns (#003040001, Diagenode).
  • a sequencing library may comprise adaptor sequences.
  • the adaptor sequences may vary depending on the sequencing method used subsequent to preparing the sequencing library. For example, where Illumina sequencing is used, i5 and i7 ends may be attached to the nucleic acid fragments.
  • the first and second DNA molecule comprise the adaptor sequences and are added by the transposase to the one or more chromatin-binding sites. This may eliminate a time-consuming step in the workflow and lower the amounts of starting material required.
  • methods as described herein for preparing a sequencing library may further comprise a step for integrating said adaptor sequences, which may be part of the amplification step.
  • obtaining a sequencing library comprises an amplification step.
  • Amplification of the sequencing libraries may be achieved by various techniques known in the art.
  • the best-known technique for nucleic acid, in particular DNA, amplification is polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, reannealed, extended, and dissociated to amplify the number of copies of the nucleic acid. This cycle can be repeated.
  • the amplification step comprises insertion of next-generation sequencing indices and/or adaptors.
  • the primers may comprise sequences hybridisable to the sequence comprised in the oligonucleotides comprised in the transposomes.
  • primers may comprise sequences necessary for sequencing.
  • specific primers are used that are compatible with the subsequently used sequencing method.
  • Illumina sequencing as one method of sequencing, is compatible with primers introducing flowcell ends, which can hybridize to the flowcell needed in cluster amplification.
  • primers may introduce i5 and i7 ends for Illumina sequencing.
  • primers may introduce barcodes for multiplexing.
  • barcodes comprised in the primer sequences may be used as unique molecular identifiers to discriminate between PCR duplicates and/or as defined barcodes to combine multiple experiments in one sequencing run.
  • the methods as described herein comprise an amplification step, it is important to achieve a sufficient amount of eluted library without overamplifying the libraries.
  • sequencing libraries are amplified for at least 18, at least 17, at least 16, at least 15, least 14, least 13, at least 12, at least 11 , at least 10, at least 9 or at least 8 cycles.
  • the cycle number may depend on the starting cell number and context. Without being bound by any particular theory, a cycle number greater than 18 may reduce the information that can be obtained from sequencing.
  • amplification is directly followed by a purification step, e.g. using AMPure beads directly after the amplification step.
  • the present method may integrate adapters into DNA in the vicinity of the transposome that is linked to a specific binding agent.
  • the exact sites of integration may be affected by the accessibility of surrounding DNA. For this reason, fragments that share exact starting and ending positions can indeed be common, and such ‘duplicates’ may not be due to duplication during PCR.
  • fragments that share exact starting and ending positions are used for further analysis according to the present method.
  • fragments that share exact starting and ending positions are disregarded in the analysis according to the present method. For example, Picard command-line tools may be used to check the duplication rate.
  • the product of amplification may be characterized by techniques such as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing.
  • the purification may be followed by a determination of the size distribution and/or concentration of libraries by methods known in the art, e.g. capillary electrophoresis using a TapeStation (Agilent Technolgies) or Fragment Analyzer (Agilent Technologies) or equivalent.
  • libraries for which the concentration is considered too low may be reamplified.
  • assessing library concentration only after a purification step in which the PCR polymerase and unique dual-indexing primers (UDI) are removed and samples are left in an elution buffer may not be optimal for potential reamplification PCR reactions.
  • samples that have been re-amplified under these conditions may be less comparable with other samples. While re-amplification is technically possible if the required reagents containing the polymerase are added again, there is a risk of over-amplification.
  • amplification may be first performed using a lower number of cycles, for instance 9 cycles for histone modifications, e.g. H3K4me3, and 12 cycles for non-histone proteins such as PRC2 proteins. Following initial amplification, the concentration of the obtained libraries may be quantified, e.g.
  • DNA standards of a known concentration may be used to create a calibration curve to calculate the concentration of the sequencing libraries.
  • a concentration of 5 nM at this step may be regarded as minimum to proceed. If the concentration is lower, additional PCR cycles may be added. This way, an appropriate number of PCR cycle may be chosen for each sample separately, ensuring the balance between a sufficient enrichment and minimizing the risks of PCR-induced artifacts and biases.
  • the quality of the sequencing library can also be evaluated by testing for enrichment at target loci. This may be done using qPCR on the ready-to-sequence libraries. Such a quality test can give an indication of the success of the methods disclosed here, while only sequencing can give a complete insight into the quality and biology of the data obtained with the disclosed methods.
  • the enrichment test may be used to decide whether to proceed with sequencing the libraries obtained or to repeat the experiment.
  • sequencing libraries generated from different samples may be mixed in an equimolar sequencing pool prior to sequencing.
  • the method as described herein involves determining the sequence of the excised and tagged DNA segment.
  • sequencing can be performed using pyrosequencing on a solid support (such as 454 sequencing, Roche), sequencing-by-synthesis with reversible terminations (such as with the ILLUMINA® Genome Analyzer), or nanopore technology (e.g. Oxford Nanopore Technologies MinlONTM).
  • the excised and tagged DNA fragments are analyzed, for example by determining the nucleotide sequence.
  • the nucleotide sequence is determined using sequencing or hybridization techniques with or without amplification.
  • sequencing may be performed using single-end sequencing. In other embodiments, sequencing may be performed using paired-end sequencing.
  • the at least one chromatin binding site of the chromatin-associated factor of interest in the cell is determined. This step may also be referred to as “mapping” of the at least one chromatin binding site of the chromatin-associated factor of interest.
  • data may be analyzed using sequence comparison software that aligns sequenced nucleic acids to genomic sequences. Genomic sequences are generally known and obtainable from freely accessible data sources.
  • a match of a sequenced nucleic acid, which is found in the sample to be analyzed, and a genomic sequence may be used as indicator that said sequenced nucleic acid is bound by the chromatin-associated factor of interest, for example the histone or transcription factor, which is recognized by the first antibody in the methods as described herein.
  • the method as described herein may involve quality control of sequencing reads, for instance using FastQC (Andrews, 2010) algorithm.
  • determining the at least one binding site involves trimming of the adaptors.
  • Algorithms for adaptor trimming are well known in the art, for instance cutadapt or trimmomatic.
  • the trimmed reads are aligned to a reference genome.
  • the reference genome may be obtained by sequencing technologies or from open sources, such as the UCSC genome browser (Rosenbloom et al., 2015). Alignment methods are well known in the art, comprising, for instance, BWA software v.0.7.5a (Li and Durbin, 2009), Bowtiel and/or Bowtie2.
  • the data may be filtered for regions blacklisted by the ENCODE project (Hoffman et al., 2013) and/or multimapping reads may be removed, for instance using samtools (Li et al., 2009). Alignment coordinates may be converted to BED format, for instance using BEDTools v.2.17 (Quinlan and Hall, 2010). Based on matching the sequenced nucleic acids to genomic sequences, statistical computational methods may be used to determine regions of significant binding to distinguish them from unspecific "background signal". Peak calling may be performed, for instance, using MACS, epic2, SEACR, GoPeaks, and/or CUT&RUNTools2.0.
  • a normalization step may be performed.
  • normalization may be based on binding sites obtained using an input sample.
  • normalization may be based on binding sites obtained using an IgG sample.
  • IgG and input samples may capture the background signal and potential unspecific off-targets peaks produced by unspecific tagmentation by the transposase. Therefore, by using either IgG or input to normalize chromatin binding sites obtained in the inventive methods only true peaks will be considered for further processing.
  • an input sample is set aside prior to the permeabilization step and subjected to method steps of activating the transposase, thereby excising a DNA segment comprising at least one chromatin binding site of a chromatin-associated factor of interest and tagging the DNA with the first and second DNA molecule; determining the sequence of the excised and tagged DNA segment; and based on the determined sequence of the excised and tagged DNA segment determining the at least one chromatin binding site of the chromatin- associated factor of interest in the cell.
  • the input sample is used for normalization in the step of determining the at least one chromatin binding site of the chromatin- associated factor of interest in the cell based on the determined sequence of the excised and tagged DNA.
  • an antibody targeting the chromatin-associated factor of interest is used as first antibody and for a second portion of the cell population IgG is used as first antibody and the sequence of the excised DNA determined for the portion of the cell population using IgG as first antibody is used for normalization in the step of determining the sequence of the excised DNA for the portion of cell population using the antibody targeting the chromatin-associated factor of interest as first antibody.
  • Normalization may be further assessed in a pipeline that quantifies peaks resulting from a Tn5 bias.
  • Pipelines known in the art comprise the NOCOA pipeline as developed by Susami et al. 2022 (Susami, K., Ikeda, S., Hoshino, Y., Hyundai, S., Minami, N., 2022. Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag). Sci. Rep. 12, 11727).
  • a method for quality control by determining the percentage of false positives which may result from off-target transposase activity comprises
  • step (iii) identifying a subset of the chromatin binding sites identified in step (ii) that does not overlap with a reference set of chromatin binding sites determined for the chromatin-associated factor of interest identified using ChlP-seq,
  • step (iv) identifying a subset of the subset of chromatin binding sites identified in step (iii) that overlaps with a reference set of chromatin sites identified using ATAC-seq;
  • step (v) determining the number of chromatin binding sites identified in step (iv) and dividing said number with the number of chromatin binding sites identified in step (ii) in order to obtain the percentage of false positives.
  • step (i) is conducted with at least two replicates.
  • step (i) is conducted at least twice with the same replicate.
  • the chromatin binding site of the chromatin-associated factor of interest is present in closed chromatin.
  • the first or primary antibody specifically binds to a chromatin-associated factor of interest in closed chromatin.
  • the first or primary antibody is an antibody against H3K27me3 or H3K9me3.
  • the identified at least one chromatin binding site may be used to further infer their biological role by correlating it to other datasets including gene-expression, genome annotation, gene ontology or other systems biology datasets.
  • CUT&Tag Cleavage Under Targets and Tagmentation
  • Example 1 Conventional CUT&Tag protocols are not able to detect several non-histone proteins
  • K-562 cell culture K-562 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, #12440046) medium supplemented with 10% heat inactivated bovine serum (FBS, ThermoFisher, #26170-043) and 1% antibiotic-antimycotic reagent (ThermoFisher, #15240096). The cells were passaged three times per week in a T-75 falcon.
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS heat inactivated bovine serum
  • ThermoFisher, #26170-043 1% antibiotic-antimycotic reagent
  • HPC7 cell culture HPC7 cells were cultured in IM DM medium supplemented with 10% serum (ES), 0.1% stem cell factor (SCF), 1% 2-Mercaptoethanol, 1% L-Glutamine & Glutamax and 1% Penicillin-Streptomycin.
  • Chromatin Immunoprecipitation HeLa cells were harvested using trypsin-EDTA and crosslinked with formaldehyde according to the iDeal ChlP-seq Kit for Histones (Diagenode). Chromatin was sheared in a Bioruptor (Diagenode) for 11 cycles (30s ON/30s OFF). Sheared chromatin stored at -80°C until use. A shearing assessment was performed according to the manufacturer’s instructions. Fragment size was assessed using Bioanalyzer (Agilent Technologies). There was a ChlP-qPCR conducted as a quality control for each new chromatin batch.
  • IP mixes were prepared according to the iDeal ChlP-seq kit for histones (Diagenode) and using antibodies against H3K4me3 and H3K9me3 (both Diagenode). Sheared chromatin was added corresponding to the desired cell number.
  • Enrichment qPCR with primer pairs of positive and negative control regions using KAPA SYBR® FAST qPCR master mix 2x (Roche) were run on LightCycler96 (Roche) and normalised using input.
  • Cut & Tag was performed as described in conventional protocols. In brief, cells were harvested, washed in wash buffer (20 mM HEPES pH 7.5; 150 mM NaCI; 0.5 mM Spermidine; 1x Protease inhibitor cocktail), and bound to activated, magnetic Concanavalin A (ConA) beads (Polysciences, #86057) for 8 min (10 pl per sample).
  • wash buffer (20 mM HEPES pH 7.5; 150 mM NaCI; 0.5 mM Spermidine; 1x Protease inhibitor cocktail
  • ConA magnetic Concanavalin A
  • Beads were resuspended in 50 pl antibody buffer per sample (20 mM HEPES pH 7.5; 150 mM NaCI; 0.5 mM Spermidine; 1x Protease inhibitor cocktail; 0.05% digitonin; 2 mM EDTA; 0.1% BSA) and 1 pg of an antibody against the protein or histone modification of interest was added and incubated overnight at 4°C. The next day, the buffer was removed and 1 pg of a secondary antibody (anti-rabbit or anti-mouse), already mixed 1 :100 in 100 pl of antibody buffer, was added and incubated for 45 min at RT. Unbound antibodies were washed away in 1 ml Digwash buffer (wash buffer containing 0.05% digitonin) per wash step.
  • 1 ml Digwash buffer wash buffer containing 0.05% digitonin
  • pA-Tn5 was added in 1 :250 Dig-300 buffer (20 mM HEPES pH 7.5; 300 mM NaCI; 0.5 mM Spermidine; 1x Protease inhibitor cocktail; 0.05% digitonin) and incubated at RT for 1h. Unbound pA-Tn5 was washed away using 1 ml of the same buffer per wash step. Beads were resuspended in 300 pl tagmentation buffer (Dig-300 buffer containing 10 mM MgCI 2 ) and the tagmentation step took place at 37°C for 1h. It was stopped by adding EDTA, SDS and proteinase K, and the following protein digestion took place at 55°C according to the manufacturer’s instructions.
  • a library quantification using the KAPA Library Quantification Kit (Roche, #07960140001) was performed. An aliquot of 0.5 - 1.0 pl was taken from the CUT&Tag samples and mixed in a 1 :1000 dilution with library dilution buffer (10 mM Tris-HCI, pH 8.0 - 8.5, 0.05% Tween® 20). These dilutions were vortexed at full speed for 10 seconds. A qPCR was conducted using the sample dilutions and DNA standards of known concentrations (20 pM to 0.0002 pM) and the samples’ concentration was calculated according to the manufacturer’s instructions.
  • the KAPA Library Quantification Kit contains primers matching to sequencing adapters (included in the kit). Therefore, the qPCR amplifies fragments which contain the Illumina sequencing primers. If necessary, the samples could be re-amplified according to the manufacturer’s instructions to reach a sufficient concentration high to fulfil the sequencing requirements, and then purified with AM Pure XP beads as described above.
  • Cutadapt removes adapter sequences from high-throughput sequencing reads.
  • Trimmed reads were aligned to the reference genome obtained from the UCSC genome browser using BWA software v.0.7.5a. Samples were filtered for regions blacklisted by the ENCODE project. Multimapping reads were removed using samtools while PCR duplicates were kept. Alignment coordinates were converted to BED format using BEDTools v.2.17. Peak calling was performed using MACS2 without the broad option, with a q-value of 1e-05. Differential binding analysis was performed with the R/Bioconductor package: DiffBi nd . Heatmaps around TSS were generated using computeMatrix functions from deepTools using the bigwig files. For the generating of screenshots of genome landscapes, the USCS genome browser and the IGV server have been used.
  • HDAC2 Diagenode, #015200201 mouse CUT&Tag, ChIP
  • Low library concentrations were observed except for SUZ12 ( Figure 1).
  • the sequencing profiles showed almost void profiles ( Figure 2) and a high percentage of unmapped reads of 40-90% per sample. These unmapped reads mapped to the E. coli genome instead, corresponding to a contamination originating from the pA-Tn5 protein.
  • E. coli peaks there were only few reads in the samples and no antibody-specific peaks.
  • even the SUZ12 samples that showed a higher library concentration did not lead to the expected results, but had more reads mapping to the E. coli reference genome than other samples.
  • the conventional CUT&Tag protocol was also performed for the histone modifications H3K4me3, H3K27me3 and H3K9me3 using 50,000 K-562 cells and for the non-histone proteins CTCF, NRF1 and HDAC2 using 50,000 or 300,000 K-562 cells.
  • ChIP was performed using the True MicroChlP-seq kit (Diagenode, #C01010132) with the same antibodies using 50,000 cells for histone modifications and the Deal ChlP-seq Kit for Transcription Factors (Diagenode, #001010055) with 4 million cells for non-histone proteins, respectively. Two replicates per condition were used for all samples.
  • a low peak score indicates a low confidence in peak calling. It is worth noting that even with ChIP using 4 million cells, the peak scores and FRiP were low for NRF1 and HDAC2 but acceptable for CTCF. This highlights the need for a reliable method to profile non-histone proteins. Nevertheless, the CUT&Tag samples for the histone modifications H3K4me3, H3K27me3 and H3K9me3 had a similar quality to their ChlP-seq counterparts, but with a lower proportion of uniquely mapped reads and a higher proportion of reads in peaks (FRiP). The latter underlines the low background signal in CUT&Tag, which can also be seen in the genome tracks ( Figure 4). It should be noted that the comparison of CUT&Tag with ChIP for some parameters, such as the number of peaks, is limited due to the differences in the data, the shape of the peaks and the high signal-to-noise ratio in CUT&Tag ( Figure 4).
  • Figure 4A showed a good overlap among CTCF CUT&Tag samples and with ChIP controls.
  • the overlaps of HDAC2 and NRF1 between replicates and the ChIP controls are limited.
  • the conventional CUT&Tag protocol could detect peaks better as shown in the overlaps with ChIP.
  • the sequencing statistics, overlap of peaks among replicates and with ChIP controls, and genome tracks confirm that conventional CUT&Tag protocols are able to detect histone modifications. For these samples, the conventional CUT&Tag protocol shows a good reproducibility according to ENCODE criteria.
  • digitonin is used to permeabilize the cells by interacting with cholesterol in the membrane, thereby allowing the antibodies, pA-Tn5 and other reagents to enter the cell membrane and nucleus.
  • Digitonin is toxic, especially when dissolved in DMSO, which penetrates the skin, and therefore requires handling under a chemical safety hood.
  • the digitonin stock solutions are not stable for long, so a new stock solution often needs to be prepared.
  • digitonin is extracted from the Digitalis purpurea plant, resulting to batch-to-batch variations. The use of different batches of digitonin in turn affects the reproducibility of the CUT&Tag samples.
  • Triton X-100 which has been used to permeabilize cells in other methods, such as CUT&RUN.
  • the aim of this example is to investigate whether the use of Triton X-100 as a permeabilization reagent in CUT&Tag provides comparable cell permeabilization and sequencing results to digitonin.
  • Table 5 Sequencing statistics of experiments using the conventional CUT&Tag protocol with either Triton X-100 (TX) or digitonin (Dig; control) as permeabilization reagent. The experiments were carried out in duplicates with 50,000 K-562 cells per experiment. FRiP: Fraction of peaks in reads.
  • Triton X-100 as a cell permeabilization reagent in CUT&Tag resulted in good overlap between replicates and compared to data obtained with digitonin. Triton X-100 therefore represents a safer and less variable alternative to digitonin for the purposes of CUT&Tag experiments.
  • a different buffer base was used. Without being bound to a particular theory, using a different buffer base may achieve a more appropriate pH range during the tagmentation step and/or chelate Mg 2+ in the solution, e.g. residual Mg 2+ from the cells, which may help to avoid premature activation of pA-Tn5 and non-specific cutting of accessible chromatin.
  • light crosslinking was performed immediately after the harvest.
  • light crosslinking e.g. using 0.1% formaldehyde for 5 min, may further stabilize protein DNA-interactions without resulting in the masking of epitopes and thus making the target protein less accessible for antibodies and pA-Tn5.
  • 0.2 ml PCR tube strips were used for each individual sample instead of 1.5 ml tubes. This may advantageously reduce the amount of beads clumping on the tube wall or cap and may facilitate resuspension, thereby increasing the recovery.
  • the volumes of buffers were adjusted accordingly, such that the volume of the wash buffers and of the tagmentation buffer was reduced to 200 pl as compared to 0.8 to 1.0 ml used in conventional protocols.
  • These changes enable the use of multichannel pipettes for the washing steps, which has the advantage of reducing the hands-on time during the washing steps, thereby increasing throughput.
  • the use of 0.2 ml PCR tubes may improve data quality and recovery as the exposure time of samples to buffers is reduced. Without being bound by theory, decreased exposure times may prevent the change of chromatin structures and detachment of proteins from DNA.
  • MgCI 2 was added prior to the activation of the transposase in the tagmentation buffer.
  • the amount of pA-Tn5 was increased from a 1 :250 to a 1:100 dilution and the amount of primary antibody was increased from 1 pg to 2 pg per reaction for 5,000 cells and more and to 3 g per reaction for 50,000 cells and more, respectively. Dilution of pA-Tn5 and antibody amounts were adjusted based on routine measures known to the skilled person.
  • the amount of pA-Tn5 and antibodies used in CUT&Tag is evaluated for each batch of pA-Tn5 and/or antibody and for each target chromatin factor of interest.
  • Methods to determine an optimized dilution of pA-Tn5 and amount of primary and secondary antibodies are well known in the art. These include, for example, the titration of pA-Tn5 and antibody amounts, respectively, and, after subjecting the samples to CUT&Tag, evaluating the library concentration and the data obtained after sequencing, e.g. by visual inspection and bioinformatic analysis.
  • K-562 cell culture K-562 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, #12440046) medium supplemented with 10% heat inactivated bovine serum (FBS, ThermoFisher, #26170-043) and 1% antibiotic-antimycotic reagent (ThermoFisher, #15240096). The cells were passaged three times per week in a T-75 falcon.
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS heat inactivated bovine serum
  • ThermoFisher, #26170-043 1% antibiotic-antimycotic reagent
  • CUT&Tag In general, all wash steps and buffer removals after the cells were bound to the magnetic ConA beads took place on a magnet after a quick spin. All incubations took place on a rotator except the tagmentation step which took place on a thermocycler for 0.2 ml tubes. A multichannel pipette can be used for most wash steps and some reagents additions. Samples were processed in duplicates or triplicates.
  • CUT&Tag an aliquot of CUT&Tag input can be put aside at this step.
  • the remaining cells were bound to ConA beads for 20 min (10 pl per sample). Beads were resuspended in 50 pl of Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1% BSA, 5 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine) per sample, distributed into 0.2 ml tube strips and 2 pg of the primary antibody for 5,000 cells and more and 3 pg of the primary antibody for 50,000 cells and more, respectively, was added unless stated otherwise.
  • Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1%
  • Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KOI, 250 mM sucrose, 150 mM NaCI, 2mM EDTA, 0.1 % BSA, 5 mM MgCI2, 1X protease inhibitor cocktails, 0.5 mM spermidine). 3 pg of the secondary antibody was added and incubated for 45 min.
  • Tagmentation Buffer D300 (20 mM Tricine KOH pH 7.8, 0.1 % Triton X-100, 25 mM KOI, 250 mM sucrose, 300 mM NaCI, 15 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine) and incubated at 37°C for 1h. Input samples were included at this step. Tagmentation was stopped by adding 0.5 M EDTA, 10% SDS and proteinase K, and the following protein digestion took place at 55°C according to the manufacturer’s instructions.
  • the KAPA Library Quantification Kit contains primers matching to sequencing adapters (included in the kit). Therefore, the qPCR amplifies fragments which contain the Illumina sequencing primers. If necessary, the samples could be re-amplified according to the manufacturer’s instructions to reach a sufficient concentration high to fulfil the sequencing requirements, and then purified with AM Pure XP beads as described above.
  • HDAC2 Diagenode, #015200201 mouse CUT&Tag, ChIP
  • 50,000 K-562 cells were subjected to either the conventional CUT&Tag protocol described in Example 1 or the CUT&Tag protocol described in this example with or without crosslinking.
  • Antibodies targeting CTCF or H3K4me3 were used as well as IgG (control).
  • a high number of reads also increases the number of peaks, as the background is artificially enriched and called as a peak. This was the case for CTCF CUT&Tag replicate B in 100,000 cells: due to the high number of reads for this replicate, some of the background signal was sufficiently enriched to be called as peak. This contributed to the higher FRiP of 28% and a much higher coverage in genome tracks, and many small enrichments were called as peaks ( Figure 8). This also showed that a too high sequencing depth may bias the results. Also, the scale of the heatmap was increased to 150, while the replicates using 50,000 cells showed a signal of about 25 on the scale ( Figure 9). Few of the false positive peaks in background were expected to overlap with true peaks of the other samples.
  • the CUT&Tag replicates showed a high correlation of around 0.9 based on peaks occupancy (Figure 8), except one CUT&Tag replicate using 100,000 cells, which also showed a much lower signal in genomic tracks and other aspects of the results.
  • the overlap between all CUT&Tag and the corresponding ENCODE ChIP reference data was only 0.2.
  • the correlation between the two ENCODE EZH2 replicates was only about 0.3, which is another indication that ENCODE data is not always a good reference.
  • the EZH2 samples also showed a high correlation between 0.7 and 0.9.
  • the correlation between CUT&Tag and ENCODE data was basically non-existent.
  • the same trend was observed for the HDAC2 CUT&Tag data.
  • the correlation was around 0.95 between the HDAC2 CUT&Tag replicates, but only 0.4 when compared to the ENCODE data. This shows that the CUT&Tag protocol disclosed herein resulted a high reproducibility between CUT&Tag replicates.
  • sucrose as an exemplary crowding agent may have stabilised the native protein conformation and proper folding by restricting the space. This may have improved the recognition of CTCF, EZH2 and HDAC2 by their specific antibodies.
  • sucrose as an exemplary crowding agent may have enhanced specific protein-protein and protein-DNA interactions, especially for proteins with a low binding affinity. Said effect may have stabilised the binding of CTCF, EZH2 and HDAC2 to their specific chromatin binding sites.
  • sucrose as an exemplary crowding agent may have increased the viscosity of the solution, which can reduce protein diffusion. This may have improved the interaction of the specific antibodies with CTCF, EZH2 and HDAC2.
  • the use of another buffer basis may have generated a more suitable pH range during the tagmentation step.
  • the use of light crosslinking immediately after the harvest may have further contributed to the improved detection.
  • lowering of the buffer volumes to 200 pl per step and per sample allowed more efficient washing and reactions.
  • EZH2 and HDAC2 the data showed a higher success than conventional protocols in HPC7 cells, or even in 300,000 of K-562 cells.
  • the comparison of CUT&Tag data with ChlP-seq data is limited due to the high background and lower sensitivity of ChlP-seq, which may lead to false negative peaks.
  • This example aims to confirm the results obtained in K-562 cell using a WT/KO comparison approach in mouse embryonic stem cells (mESCs).
  • a KO in the SUZ12 gene is not only expected to decrease the signal obtained with antibodies against SUZ12 itself, but also when using antibodies against the other PRC2 core proteins EZH2 and EED as well as H3K27me3, since PRC2 is its deposition complex.
  • mESCs mouse embryonic stem cells
  • a WT/KO comparison may be achieved for all these proteins.
  • the data obtained with Suz12 _/ - cells may allow a more in-depth analysis of the WT data. For example, the lists of detected genes in WT and KO cell lines can be compared to estimate overlaps and the nature of the signal, which may be used to further improve the protocol disclosed herein.
  • Example 3 The CUT&Tag protocol disclosed in Example 3 was performed using 50,000 WT and Suz12 -/ - mESCs, generated using a CRISPR/Cas9 approach, respectively. As a KO- independent control, CTCF was assessed using CUT&Tag in both WT and Suz12 _/ - mESCs.
  • the cells were crosslinked immediately after harvest and cryopreserved. In contrast to the previous Example, the cell nuclei were not extracted, but the cells were permeabilized with Buffer D and bound to ConA beads. This may lead to a higher recovery, as several centrifugation steps are omitted compared to nucleus extraction.
  • H3K27me3 CUT&Tag experiments were performed with antibodies against H3K27me3, CTCF, EZH2, SUZ12 or IgG using 50,000 WT and Suz12 _/ - mESCs, respectively.
  • the H3K27me3 samples were processed in duplicate, the PRC2 samples in triplicate and the CTCF and IgG samples as single samples.
  • KO-only peaks were found less frequently in promoters, the classical target sites of SUZ12, but more frequently in genes (26.5 %) and intergenic regions (28.5 %), compared to the WT- only peaks with 18.6 % and 17.8 %, respectively ( Figure 14C).
  • the peaks in actively expressed regions may be due to a Tn5 bias in the protein A-Tn5 transposase used in CUT&Tag.
  • Solitary Tn5 tends to cut in accessible chromatin and generates libraries with attached sequencing adaptors. This is exploited in ATAC-seq to obtain information about accessible chromatin.
  • pA-Tn5 (comprising a still inactive Tn5) is bound to antibodies against the desired chromatin protein via the protein A part.
  • Example 3 The protocol disclosed in Example 3 was able to detect the peaks and associated genes expected for each chromatin-associated target factor and cell model. However, a bias towards open chromatin was also evident, most likely due to off-target activity of Tn5, leading to unexpected peaks at expressed genes in addition to peaks at PRC2 targets. To solve this issue, the protocol was further modified.
  • the amount of antibody was reduced from 3 pg to 1 pg per reaction. Without being bound to particular theory, this may reduce Tn5 off-target activity.
  • MgCI 2 was not added until the tagmentation step, as described in the conventional protocol. Without being bound to a particular theory, this may reduce Tn5 off-target activity and thus reduce the detection of false-positive peaks in the data set while not affecting the detection of the true positive binding sites of the chromatin-associated factor of interest as observed in Example 3.
  • peaks were normalized either using an input sample-based normalization strategy as described herein or an IgG-based normalization strategy. Without being bound to particular theory, peak normalization of CUT&Tag data may leave only true positive peaks for further processing.
  • Example 3 aims to demonstrate that further modifications to the protocol disclosed in Example 3 further improved the avoidance of false-positive peaks due to a Tn5 bias. Moreover, this example aims to demonstrate that the protocol disclosed herein can be successfully applied using cell numbers of 5,000 cells even when studying non-histone proteins.
  • ES-E14TG2a Culture of mouse embryonic stem cells (mESCs).
  • ES-E14TG2a were cultured in Glasgow's Minimal Essential Medium (GMEM, Gibco, 11710035), supplemented with 20% FBS (ES cell qualified, FisherScientific, #15946942), 1% Glutamax (FisherScientific, #11574466), 1% non-essential amino acids (FisherScientific #12084947), 1 mM sodium pyruvate (FisherScientific, #11530396), 1 mM 2-Mercaptoethanol (ThermoFisher, #21985023), 100 II Penicillin-Streptomycin (ThermoFisher, #15140122), 1,000 ll/rnl mouse Leukaemia Inhibitory Factor (mLIF, Merck Millipore, #ESG1106), on flasks coated with 0.1% gelatine.
  • GMEM Glasgow's Minimal Essential Medium
  • FBS ES cell qualified
  • CUT&Tag In general, all wash steps and buffer removals after the cells were bound to the magnetic ConA beads took place on a magnet after a quick spin. All incubations took place on a rotator except the tagmentation step which took place on a thermocycler for 0.2 ml tubes. A multichannel pipette can be used for most wash steps and some reagents additions. Samples were processed in duplicates or triplicates.
  • Cells were crosslinked in 0.1% formaldehyde (Merck Millipore, #F8775) for 5 min and quenched with glycine for additional 5 min. Cells were washed twice in Dulbecco's phosphate-buffered saline (DPBS, ThermoFisher, #14040133). From this point, all steps took place on ice or 4°C. Cells were resuspended in 50 pl of cold Buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, sucrose 250 mM, 5 mM MgCI 2 , 1X protease inhibitors cocktail) per sample.
  • DPBS Dulbecco's phosphate-buffered saline
  • CUT&Tag an aliquot of CUT&Tag input can be put aside at this step.
  • the remaining cells were bound to ConA beads for 20 min (10 pl per sample).
  • Beads were resuspended in 50 pl of Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1 % BSA, 1X protease inhibitor cocktail, 0.5 mM spermidine) per sample, distributed into 0.2 ml tube strips and 1 pg of the primary antibody was added unless stated otherwise. This was incubated overnight at 4°C. The next day, the buffer was removed.
  • Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1 % BSA, 1X proteas
  • Beads were again resuspended in 100 pl of Antibody buffer D (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1 % BSA, 1X protease inhibitor cocktail, 0.5 mM spermidine). 1 pg of the secondary antibody was added and incubated for 45 min.
  • Tagmentation Buffer D300 (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 300 mM NaCI, 15 mM MgCI 2 , 1X protease inhibitors cocktail, 0.5 mM spermidine) and incubated at 37°C for 1h. Input samples were included at this step. Tagmentation was stopped by adding 0.5 M EDTA, 10% SDS and proteinase K, and the following protein digestion took place at 55°C according to the manufacturer’s instructions.
  • the KAPA SYBR® FAST qPCR Master Mix (2X) contains primers matching to sequencing adapters (included in the kit). Therefore, the qPCR amplifies fragments which contain the Illumina sequencing primers. If necessary, the samples could be re-amplified according to the manufacturer’s instructions to reach a sufficient concentration high to fulfil the sequencing requirements, and then purified with AM Pure XP beads as described above. Table 10: Antibodies and enzymes used in CUT&Tag and ChlP.
  • Enrichment qPCR and the used primers After the CUT&Tag protocol disclosed herein was completed, a qPCR targeting expected loci was performed. For this, 5 pl of 1 :10 dilutions of the CUT&Tag samples were mixed with 10 pl of 2x KAPA SYBR FAST (Merck Millipore, #KK4601), 1 pl of the desired primer pair solution containing 5 mM per primer, and 4 pl of ultrapure water. The following PCR program was applied (Table 11):
  • Table 12 List of primer pairs used in CUT&Tag and Ch IP
  • Samples were purified using DiaPure columns (500 pl Binding buffer was added per sample). Optionally, between the two wash steps, one step of adding 1 pl of RNAse directly onto the filter for 15 minutes was added. Samples were then eluted in 30 pl of elution buffer. The samples were quantified by Qubit and kept until the tagmentation step at +4°C. Optionally, the samples were further quantified by FA/FEMTO with a gDNA kit. For the tagmentation step, 270 pl of tagmentation buffer with pA-Tn5 diluted 1 :100 (calculated as in total volume of 300 pl) per sample was added and incubated in a thermomixer at 37°C for 1 hour, set at 800 rpm. The tagmentation reaction was stopped and the samples were further treated as described for conventional CUT&Tag.
  • Bioinformatics analysis of CUT&Tag Quality control of sequencing reads was performed using FastQC. Trimming of the adaptors was performed with cutadapt. Trimmed reads were aligned to the reference genome (mm 10) obtained from the LICSC genome browser using BWA software v.0.7.5a. Samples were filtered for regions blacklisted by the ENCODE project.
  • Multimapping reads were removed using samtools (Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009.
  • samtools Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009.
  • SAMtools Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009.
  • SAMtools Li, H., Handsaker
  • the CUT&Tag protocol as described herein was performed using two different antibodies against H3K27me3 (Diagenode, #C15410195; Merck Millipore, #07-449), CTCF, SLIZ12 and IgG in fresh WT mESCs and Suz12 _/ - mESCs, each in duplicates, except CTCF and IgG.
  • H3K27me3 Diagenode, #C15410195; Merck Millipore, #07-449
  • CTCF SLIZ12 and IgG
  • SLIZ12 and IgG fresh WT mESCs
  • Suz12 _/ - mESCs each in duplicates, except CTCF and IgG.
  • One input sample was processed per cell model. 50,000 freshly harvested cells were used per reaction.
  • T o quantify the T n5 bias, a NOCOA (peaks not overlapping with Chi P, but overlapping with ATAC-seq) pipeline described by Susami et al., 2022 (Susami, K., Ikeda, S., Hoshino, Y., Hyundai, S., Minami, N., 2022. Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag). Sci. Rep. 12, 11727), although here, only peaks in common among replicates were used in the analysis.
  • Genome tracks at the HOXA cluster revealed successful detection of SUZ12 and H3K27me3 independent of the antibody used ( Figure 21). All samples in WT mESC cells showed the expected pattern at the HOXA cluster. The signal of SUZ12 samples obtained using CUT&Tag as described herein were even twice as high as for the ChIP reference data. As expected, none of the SLIZ12 KO samples showed enrichment and thus, no peak.
  • H3K27me3 and SLIZ12 peaks obtained for wt mESCs were plotted on their respective set of target genes (Figure 22A).
  • the KO signal was found to be similar to the IgG signal.
  • Both antibodies against H3K27me3 showed similar signal intensity in WT cells. Taken together, this indicates a strong positive signal in WT samples and mere background signal in KO samples at these sites.
  • a strong overlap between the peaks of the H3K27me3 repeats was obtained with Merck Millipore's antibody and normalized with IgG (Figure 22B). Boxplots of the peaks’ sites shows that the signal at the common, but also at the specific sites is of a similar intensity ( Figure 22C).
  • the CUT&Tag protocol disclosed herein showed a high reproducibility in a large number of loci for all samples when using low cell number of 5,000 mESCs. Peak calling may be further optimized as described in Example 6 below.
  • ChlP-seq The most commonly used peak callers for chromatin profiling are MACS2 and epic2. However, these were developed and optimized for ChlP-seq. While ChlP-seq and CUT&Tag are similar in their basic principles, the differences in sample preparation and fragmentation mechanisms may require different approaches for peak calling. ChlP-seq often results in high background levels caused by cross-linking and shearing as well as high sequencing depth. Peak callers developed for ChlP-seq often rely on background models to account for these high background levels and detect antibody-specific enrichment against this background. In contrast, the chromatin fragments excised by Tn5 transposase result in narrower, more distinct peaks and a much lower background signal.
  • the Sparse Enrichment Analysis for CUT&RUN was developed for CUT&RUN and uses the genome-wide background noise as calibration. This enables accurate differentiation between true and false-positive peaks.
  • GoPeaks was developed for CUT&Tag and was suggested to improve peak detection in variable peak profiles compared to GoPeaks, especially when narrow peaks, as observed for H3K4me3 and transcription factors, are used as input data.
  • the CUT&RUNTools2.0 were developed to analyze CUT&RUN and CUT&Tag data at the single cell and bulk level using an end-to-end pipeline with MACS2 for the peak calling step.
  • CUT&Tag samples obtained for H3K27me3, or IgG as control, in mES WT cells and in Suz12- cells using the CUT&Tag protocol disclosed herein were analyzed using to different peak callers and settings without or upon normalization using IgG (Table 13). For GoPeaks, all results shown used IgG normalization as this is required for this peak caller. Table 13: CUT&Tag samples used in different peak calling algorithms and settings.
  • Table 14 Proportion of shared peaks between H3K27me3 replicates in WT mESCs with or without IgG normalization.
  • Optimal peak calling parameters are expected to detect only a few peaks in IgG and MTF2 KO samples, but be able to detect true MTF2 peaks in DM SO-treated control cells that resemble WT conditions.
  • the first test was performed with the default settings of each peak caller (Table 16).
  • MLL-AF9 is a fusion protein resulting from a translocation of chromosomes 9 and 11 in humans and 4 and 9 in mice. When expressed in hematopoietic stem and progenitor cells (HSPCs), it can lead to acute myeloid leukemia (AML) in humans and mice. Therefore, MLL- AF9 has been extensively studied in the context of leukemia development in mouse models. Previous research has shown that MLL-AF9 leads to gene activation through the abnormal recruitment of DOT1-like histone H3K79 methyl transferase and SEC.
  • MLL-AF9 causes upregulation of HOXA9 and EZH2 expression, among others, which promotes proliferation while inhibiting differentiation and apoptosis through Cdkn2a repression. While MLL-AF9’s activity in hematological differentiation has been described, little is known about its role in embryonic differentiation is.
  • this example aims to investigate the role of the oncogenic fusion protein MLL-AF9 in mESCs during their development to embryonic bodies and the interaction of MLL-AF9 with Polycomb repressive complexes (PRC).
  • PRC Polycomb repressive complexes
  • embryonic stem cells i.e. three-dimensional structures containing cells from the three germ layers (mesoderm, endoderm and ectoderm), was chosen as a model because it resembles the differentiation of mESC in vivo.
  • embryoid bodies i.e. three-dimensional structures containing cells from the three germ layers (mesoderm, endoderm and ectoderm)
  • Differentiation of embryoid bodies allows a comprehensive study of cell differentiation and tissue development and provides insights into the role of relevant factors in differentiation in a controlled in vitro environment, thus providing a physiologically relevant model to study MLL-AF9 in embryonic differentiation.
  • ES-E14TG2a Culture of mouse embryonic stem cells (mESCs).
  • ES-E14TG2a were cultured in Glasgow's Minimal Essential Medium (GMEM, Gibco, 11710035), supplemented with 20% FBS (ES cell qualified, FisherScientific, #15946942), 1 % Glutamax (FisherScientific, #11574466), 1 % non-essential amino acids_(FisherScientific #12084947), 1 mM sodium pyruvate (FisherScientific, #11530396), 1 mM 2-Mercaptoethanol (ThermoFisher, #21985023), 100 II Penicillin-Streptomycin (ThermoFisher, #15140122), 1 ,000 ll/rnl mouse Leukaemia Inhibitory Factor (mLIF, Merck Millipore, #ESG1106), on flasks coated with 0.1% gelatine.
  • GMEM Glasgow's Minimal Essential Medium
  • EBs were collected by transferring the cell suspension into 15 ml tubes and letting it sit for 3 min. The supernatant was discarded. The cell pellet was washed with 10 ml PBS. 1 ml trypsin-EDTA was added and incubated at 37°C for 5 min. Cells were aspirated in 9 ml IMDM+10% FBS and released towards the tube wall. Cells were centrifugated at 180 x g for 5 min and the supernatant was discarded. Cells were resuspended in 1 ml IMDM+10%FBS, then additional 4 ml were added. The cells were passed through a cell strainer of 40 pm and counted. Samples for CUT&Tag, ATAC-seq and RNA-seq were taken at day 0 (mESC) and day 5 (mEB).
  • ATAC-seq was performed on samples of mESC and mEB. All cell conditions (Table 17) were processed in duplicates. Table 17 List of cell conditions processed with ATAC-seq and RNA-seq 0330] ATAC-seq samples were processed using the ATAC-seq kit (Diagenode, #C01080002) and following the manufacturer’s instructions. Quality was checked on Fragment Analyzer using the NGS kit (Agilent Technologies, #DNF-473-0500). Sequencing was performed in paired-end 50 bp on an Illumina NovaSeq6000 flow cell. Quality control of sequencing reads was performed using FastQC (Andrews, 2010).
  • Trimming of the adaptors was performed with Trim_Galorel. Trimmed reads were aligned to the reference genome (mm 10) obtained from the UCSC genome browser (Rosenbloom et al., 2015) using Bowtie2. Samples were filtered for regions blacklisted by the ENCODE project (Hoffman et al., 2013). PCR duplicates and multimapping reads were removed using samtools (Li et al., 2009). Alignment coordinates were converted to BED format using BEDTools v.2.17 (Quinlan and Hall, 2010) and peak calling was performed using MACS2 (Zang et al., 2009).
  • RNA-seq was performed on samples of mESC and mEB. Each cell condition was processed in duplicates. First, total RNA was extracted of the samples using the miRNeasy mini kit (Qiagen, #217004) and following the manufacturer’s instructions. Ribosomal RNA has been depleted using the NEBNext rRNA Depletion Kit v2 (New England Biolabs, #E7400).
  • the D-Plex Total RNA-seq Kit Total RNA library preparation kit for Illumina® sequencing, Diagenode, #C05030031
  • Total RNA library preparation kit for Illumina® sequencing Diagenode, #C05030031
  • JJsing this approach not only mRNA, but also noncoding RNAs are captured. However, for the further processing of the data in the multi-omics analysis, only mRNAs were included.
  • the noncoding RNAs could be processed and integrated in a future project using the MGCount tool (Hita et al., 2022). Sequencing of the samples was performed on an Illumina NovaSeq6000 instrument producing 50 bp paired-end reads running Control Software 1.7.0.
  • RNA abundance was estimated with MGCount (Hita et al., 2022). Further analysis consider only features with counts per million (CPM) above 5 for at least 6 samples.
  • RNA-seq solely protein-coding genes were considered for the analysis. Additionally, genes not reaching a counts per million (CPM) expression above 5 for at least 2 samples were filtered out. For CUT&Tag and ATAC- seq, peaks called at least in both replicates of any sample type were considered for the analysis. Subsequently, following DiffBind pipeline, overlapping peaks were recentred at the summit and homogenised to 400 bp window length symmetrically defined around the submit. The number of_alignments was then quantified on the recentred peaks for all samples (samples where peak was not called included) resulting into the enrichment matrix.
  • CPM counts per million
  • MLL-AF9 a marker for hematopoietic development also known as Kdr and VEGFR-2, as well as Brachyury and Eomes, were found to be downregulated compared to WT cells.
  • both the WT and the two MLL-AF9 clones showed a comparable gene expression profile and similar chromatin accessibility at day 0 of the protocol ( Figure 38A and B, left panels).
  • the WT cells begin to express lineage-specific genes while reducing the expression of sternness-related genes, resulting in a different pattern observed in both ATAC-seq and mRNA- seq data ( Figure 38A and, right panel).
  • the clones expressing the MLL-AF9 fusion protein showed a different pattern during the EB differentiation process that more closely resembles their ESC state in addition to other changes (Figure 38B).
  • the same trend was observed for chromatin accessibility, which is consistent with open chromatin being correlated with gene expression.
  • Pim2 upregulated in the MLL-AF9 clones
  • a proto-oncogene with serine/threonine kinase activity that is involved in cell survival and proliferation and is a known inhibitor of apoptosis
  • pro-oncogenes become oncogenes and can lead to cancer development. It has been described to stimulate growth factor-independent proliferation through the phosphorylation of cell cycle regulators, e.g. CDKN1A and CDKN1 B.
  • PIM2 has been shown to stabilize p21, which despite its inhibitory effect on cell cycle progression has also been shown to inhibit apoptosis, promote proliferation and is upregulated in various cancers (Wang et al., 2010).
  • Another gene found to be upregulated was Enpp2. It was shown to be involved in cell proliferation and promotes tumor progression in multiple myeloma (Li et al., 2022).
  • expression of some developmental genes such as Handl, Hand2 and Kdrwas found to be upregulated during differentiation of WT mESCs into EBs, but not (yet) in MLL-AF9 clones.
  • Cadherin-4 (also known as R-cadherin) is a cell adhesion protein of the cadherin protein family. While cadherin-1 (E-cadherin) and cadherin-2 (N-cadherin) from the same family are known for their role in epithelial-mesenchymal transition (EMT), a normal developmental process that, when disrupted, is critical for metastasis (Loh et al., 2019), cadherin-4 is less well known.
  • EMT epithelial-mesenchymal transition
  • Binding of RYBP was gained at the Cacnalc gene. Furthermore, it was found to be associated with a more condensed chromatin and a lower expression according to ATAC-seq and mRNA-seq data ( Figure 43A). Cacnalc is known to be part of the voltage-gated calcium channel regulating blood pressure (Fu et al., 2011; Moosmang, 2003). Furthermore, binding of RYBP and RING1B was gained at Zfp442, a gene encoding for a zinc finger protein with unknown function. As most zinc finger proteins comprise a DNA binding capacity, such activity is also expected for ZFP442.
  • Binding of SLIZ12 was observed to be gained and lost at similar genes as PRC1 proteins (Figure 44A). These include Glis3 in D1C clone and Bcas3 in most samples as well as Zfp442, Cacna 1c, but not Esrrb.
  • MLL-AF9 MLL-AF9-mediated recruitment
  • MLL-AF9 was reported to recruit DOT 1 L to a set of target genes where DOT 1 L deposits H3K79me2, thereby activating these target genes (Ottersbach et al., 2018). Consistent with this mechanism, H3K79me2 was found on several upregulated genes.
  • the downregulated genes found in this project could be the result of an MLL-AF9-directed recruitment of PRC1 and PRC2 as previously described in HSCPs and/or the result of other MLL-AF9-induced changes in the cells.
  • PRC2 was found to bind Hdac4, whose resulting repression can cause inactivation of its target genes.
  • binding of MLL-AF9 and binding of some PRC1/2 proteins were associated with deregulation of some genes that are likely inhibiting and/or likely causing inhibition of differentiation into embryoid bodies and promoting proliferation, possibly leading to transformation into cancer cells when they are differentiated for a longer period of time.
  • Example 8 investigates the use of glycerol as the crowding agent. The method is performed with two reaction volumes, 0.2 ml and 1 ml. Materials and methods:
  • K-562 cell culture K-562 cells are cultured in Iscove's Modified Dulbecco's Medium (IMDM, #12440046) medium supplemented with 10% heat inactivated bovine serum (FBS, ThermoFisher, #26170-043) and 1% antibiotic-antimycotic reagent (ThermoFisher, #15240096). The cells are passaged three times per week in a T-75 falcon.
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS heat inactivated bovine serum
  • ThermoFisher, #26170-043 1% antibiotic-antimycotic reagent
  • CUT&Tag In general, all wash steps and buffer removals after the cells are bound to the magnetic ConA beads take place on a magnet after a quick spin. All incubations take place on a rotator except the tagmentation step which takes place on a thermocycler for 0.2 ml tubes or 1 .5 ml tubes. A multichannel pipette can be used for most wash steps of the 0.2 ml tubes and some reagents additions. A single-channel pipette can be used for wash steps of the 1.5 ml tubes and some reagents additions. Samples are processed in duplicates or triplicates.
  • Cells are crosslinked in 0.1 % formaldehyde (Merck Millipore, #F8775) for 5 min and quenched with glycine for additional 5 min. Cells are washed twice in Dulbecco's phosphate-buffered saline (DPBS, ThermoFisher, #14040133). From this point, all steps take place on ice or 4°C.
  • DPBS Dulbecco's phosphate-buffered saline
  • Cells are resuspended in 50 pl of cold Antibody buffer D-control (20 mM Tricine KOH pH 7.8, 0.1 % Triton X-100, 25 mM KCI, 5 mM MgCI 2 , 1x protease inhibitors cocktail), Antibody buffer D-sucrose (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, sucrose 250 mM, 5 mM MgCI 2 , 1x protease inhibitors cocktail) or Antibody buffer D- glycerol (20 mM Tricine KOH pH 7.8, 0.1 % Triton X-100, 25 mM KCI, glycerol 250 mM, 5 mM MgCI 2 , 1x protease inhibitors cocktail) per sample. If desired, an aliquot of CUT&Tag input can be put aside at this step. The remaining cells are bound to ConA beads for 20 min (10 pl per sample).
  • Beads are resuspended in 50 pl (for 0.2 ml tubes) or 250 pl (for 1.5 ml tubes) of Antibody buffer D-control (20 mM Tricine KOH pH 7.8, 0.1 % Triton X-100, 25 mM KCI, 150 mM NaCI, 2 mM EDTA, 0.1 % BSA, 5 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine), Antibody buffer D-sucrose (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 150 mM NaCI, 2 mM EDTA, 0.1 % BSA, 5 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine) or Antibody buffer D-glycerol (20 mM Tricine KOH pH 7.8, 0.1% Triton X- 100, 25 mM Tricine
  • 3 pg of the secondary antibody is added and incubated for 45 min. 100 pl for 0.2 ml tubes or 500 pl for 1.5 ml tubes of a 1 :100 pA-Tn5 dilution (Diagenode, #C01070001) in Buffer D300-control (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 300 mM NaCI, 5 mM MgCI2, 1X protease inhibitors cocktail, 0.5 mM spermidine), in Buffer D300-sucrose (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 300 mM NaCI, 5 mM MgCI2, 1X protease inhibitors cocktail, 0.5 mM spermidine) or in Buffer D300-glycerol (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI
  • Tagmentation Buffer D300-control (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 300 mM NaCI, 15 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine), Tagmentation Buffer D300-sucrose (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM sucrose, 300 mM NaCI, 15 mM MgCI 2 , 1X protease inhibitor cocktail, 0.5 mM spermidine) or Tagmentation Buffer D300-glycerol (20 mM Tricine KOH pH 7.8, 0.1% Triton X-100, 25 mM KCI, 250 mM glycerol, 300 mM NaCI, 15 mM
  • Tagmentation is stopped by adding 0.5 M EDTA, 10% SDS and proteinase K, and the following protein digestion takes place at 55°C according to the manufacturer’s instructions.
  • a phenolchloroform extraction with subsequent ethanol precipitation is performed and DNA is dissolved in 10 mM Tris-HCI pH 8 1 mM EDTA.
  • the resulting DNA solution is used in a PCR with NEBNext High-Fidelity 2x PCR master mix (New England Biolabs, #M0541) and barcoded i5 and i7 sequencing primers. Samples is subjected to PCR (Table 19).
  • the KAPA Library Quantification Kit contains primers matching to sequencing adapters (included in the kit). Therefore, the qPCR amplifies fragments which contain the Illumina sequencing primers. If necessary, the samples are re-amplified according to the manufacturer’s instructions to reach a sufficient concentration high to fulfil the sequencing requirements, and then purified with AM Pure XP beads as described above.
  • CUT&Tag samples are sequenced in 2x50 bp on an Illumina NovaSeq 6000 flow cell, running NovaSeq Control Software 1.7.5, RTA v3.4.4 and bcl2fastq 2.20 v2.20.0.422.
  • Bioinformatics analysis of CUT&Tag Quality control of sequencing reads is performed using FastQC. Trimming of the adaptors is performed with cutadapt. Trimmed reads are aligned to the reference genome obtained from the UCSC genome browser using BWA software v.0.7.5a (Rosenbloom, K.R., Armstrong, J., Barber, G.P., Casper, J., Clawson, H., Diekhans, M., Dreszer, T.R., Fujita, P.A., Guruvadoo, L., Haeussler, M., Harte, R.A., Heitner, S., Hickey, G., Hinrichs, A.S., Hubley, R., Karolchik, D., Learned, K., Lee, B.T., Li, C.H., Miga, K.H., Nguyen, N., Paten, B., Raney, B.J., Smit
  • Differential binding analysis is performed with the R/Bioconductor package: DiffBind.
  • Heatmaps around TSS are generated using computeMatrix functions from deepTools using the bigwig files.
  • the USCS genome browser and the IGV server are used.
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprising:
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule;
  • the Good’s buffer is selected from Tricine, MES, ADA, PIPES, ACES, MPOSO, Cholamine chloride, MOPS, BES, TES, DIPSO, TASO, Acetamidoglycine, POPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tris, Glycinamide, Glycylglycine, Bicine and TAPS, optionally wherein the Good’s buffer is Tricine. 4. The method of item 1, wherein the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed at a salt concentration of
  • chromatin-associated factor of interest is a nonhistone protein, optionally a transcription factor or a histone reader protein.
  • the crowding agent is selected from a group comprising sucrose, hexylene glycol and glycerol, optionally wherein the crowding agent is sucrose.
  • step (iii) identifying a subset of the chromatin binding sites identified in step (ii) that does not overlap with a reference set of chromatin binding sites determined for the chromatin-associated factor of interest identified using ChlP-seq,
  • step (iv) identifying a subset of the subset of chromatin binding sites identified in step (iii) that overlaps with a reference set of chromatin sites identified using ATAC-seq;
  • step (v) determining the number of chromatin binding sites identified in step (iv) and dividing said number with the number of chromatin binding sites identified in step (ii) in order to obtain the percentage of the at least one chromatin binding site resulting from off-target activity of the transposase.
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule;
  • activating the transposase thereby excising a DNA segment comprising at least one chromatin binding site of a chromatin-associated factor of interest and tagging the DNA with the first and second DNA molecule;
  • a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell comprising the following steps:
  • transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule
  • the Good’s buffer is selected from Tricine, MES, ADA, PIPES, ACES, MPOSO, Cholamine chloride, MOPS, BES, TES, DIPSO, TASO, Acetamidoglycine, POPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tris, Glycinamide, Glycylglycine, Bicine and TAPS, optionally wherein the Good’s buffer is Tricine.
  • the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed at a salt concentration of ⁇ 300 mM NaCI.
  • any of items 16-21 wherein prior to the permeabilization step the cells are subjected to mild cross-linking conditions, optionally wherein the mild cross-linking conditions are 0.1 % formaldehyde for ⁇ 15 min, ⁇ 10 min or ⁇ 5 min.
  • the method of any of items 16-24 wherein the number of cells is ⁇ 100,000, optionally ⁇ 50,000, ⁇ 10,000, ⁇ 5,000 or ⁇ 1 ,000.
  • any of items 16-25 wherein the cell is permeabilized using Triton-X-100, optionally at a concentration of 0.1%.
  • the crowding agent is selected from a group comprising sucrose, hexylene glycol and glycerol, optionally wherein the crowding agent is sucrose.
  • step (iii) identifying a subset of the chromatin binding sites identified in step (ii) that does not overlap with a reference set of chromatin binding sites determined for the chromatin-associated factor of interest identified using ChlP-seq,
  • step (iv) identifying a subset of the subset of chromatin binding sites identified in step (iii) that overlaps with a reference set of chromatin sites identified using ATAC-seq;
  • step (v) determining the number of chromatin binding sites identified in step (iv) and dividing said number with the number of chromatin binding sites identified in step (ii) in order to obtain the percentage of false positives resulting from off- target activity of the transposase.
  • Method for generating a normalization control suitable for use in the method of item 16 or any of items 19 to 33 comprising the following steps: purifying DNA from the cell; contacting the purified DNA with a transposome that is linked to a specific binding agent suitable for binding a first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; activating the transposase; and determining the sequence of the excised and tagged DNA.

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Abstract

La présente invention concerne un procédé pour déterminer au moins un site de liaison à la chromatine d'un facteur associé à la chromatine d'intérêt dans une cellule, comportant les étapes suivantes : (i) perméabilisation de la cellule ; (ii) mise en contact de la cellule perméabilisée avec un premier anticorps qui se lie spécifiquement au facteur d'intérêt associé à la chromatine et avec un deuxième anticorps qui se lie spécifiquement au premier anticorps ; (iii) mise en contact de la cellule perméabilisée avec un transposome lié à un agent de liaison spécifique qui se lie spécifiquement au premier et/ou au deuxième anticorps, le transposome comportant une transposase et une première et une deuxième molécules d'ADN ; (iv) activation de la transposase, de sorte à exciser un segment d'ADN comportant au moins un site de liaison à la chromatine d'un facteur d'intérêt associé à la chromatine et à marquer l'ADN à l'aide de la première et de la deuxième molécule d'ADN ; (v) établissement de la séquence du segment d'ADN excisé et marqué ; et (vi) sur la base de la séquence déterminée du segment d'ADN excisé et marqué, établissement de l'au moins un site de liaison à la chromatine du acteur d'intérêt associé à la chromatine dans la cellule ; les étapes de mise en contact de la cellule perméabilisée avec le transposome et d'activation de la transposase étant effectuées en présence d'un agent d'encombrement.
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