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WO2025237407A1 - Procédés d'analyse épigénomique spatiale dans des cellules individuelles d'échantillon de tissu - Google Patents

Procédés d'analyse épigénomique spatiale dans des cellules individuelles d'échantillon de tissu

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Publication number
WO2025237407A1
WO2025237407A1 PCT/CN2025/095434 CN2025095434W WO2025237407A1 WO 2025237407 A1 WO2025237407 A1 WO 2025237407A1 CN 2025095434 W CN2025095434 W CN 2025095434W WO 2025237407 A1 WO2025237407 A1 WO 2025237407A1
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fragments
situ
cells
dna fragments
genomic dna
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Inventor
Wenyang Dong
Xiaoliang Xie
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Beijing Changping Laboratory
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Beijing Changping Laboratory
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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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present disclosure generally relates to systems, methods and compositions for analyzing genomic DNA of single cells within a tissue sample.
  • genomic DNA can also be spatially profiled using custom deterministic barcoding microfluidics, by which spatially-barcoded oligos were delivered and ligated to genomic DNA fragments inside nucleus (Deng, Yanxiang, et al. "Spatial profiling of chromatin accessibility in mouse and human tissues. " Nature 609.7926 (2022) : 375-383. ) .
  • aspects of the present disclosure are directed to methods for analyzing genomic DNA of a single cell. Aspects of the present disclosure are directed to methods for analyzing DNA of each of a plurality of single cells of a tissue sample with single cell resolution. Aspects of the present disclosure are directed to methods for the in situ spatial analysis of genomic DNA of a plurality of single cells of a tissue sample with single cell resolution.
  • Methods of the present disclosure utilize in situ methods of (1) in situ DNA fragmentation, such as transposition methods and other methods known to those of skill in the art and described herein, (2) in situ transcription methods within a single cell or within each cell of a plurality of single cells (such as in a tissue sample) to produce RNA transcripts carrying genomic DNA information.
  • the RNA transcripts of a single cell can be captured by contacting the RNA transcripts with a capture oligonucleotide, capture oligonucleotide probe or oligonucleotide capture probe as those terms are used interchangeably herein.
  • An exemplary capture oligonucleotide or capture oligonucleotide probe or oligonucleotide capture probe is a capture oligonucleotide including a poly (T) sequence, i.e. oligo (dT) sequences (which may be referred to herein as a “poly (T) oligonucleotide” or “poly (T) oligonucleotide capture probe” ) .
  • An exemplary capture oligonucleotide probe is a capture oligonucleotide including a random primer or a probe targeting a specific sequence.
  • RNA transcripts of the single cell can then be reverse transcribed into first strand complementary DNA (cDNA) .
  • cDNA first strand complementary DNA
  • the cDNA can then be barcoded.
  • the cDNAs can then be used as template to perform cDNA second-strand synthesis.
  • the cDNA can then be analyzed.
  • the cDNA can be amplified.
  • the cDNA can be sequenced.
  • the cDNA can be amplified and then sequenced.
  • the sequenced cDNA can be analyzed to provide genomic information on the single cell level, i.e. with single cell resolution. For probes targeting specific sequences, they can be amplified and sequenced to provide genomic information.
  • the method can be practice on a plurality of cells of a tissue sample, thereby providing genomic information on the single cell level, i.e. with single cell resolution, of the plurality of cells of the tissue sample and also providing spatial information.
  • the method of DNA fragmentation such as transposition, transcription and polyadenylation can be practiced in situ on cells of a tissue sample contacting a substrate having capture oligonucleotides position thereon.
  • the cells and/or cell nuclei can be permeabilized and the polyadenylated RNA can contact the substrate and be captured and reverse transcribed by the capture oligonucleotide probes with barcodes on the substrate thereby being representative of the spatial positioning of the cells of the tissue sample.
  • spatial and genomic information can be obtained from the barcoded cDNA of the polyadenylated RNA from the tissue sample, insofar as the location of the barcodes on the substrate corresponds to the location of the cell (of the tissue sample) .
  • the method of DNA fragmentation such as transposition and transcription can be practiced in situ on cells of a tissue sample contacting a substrate having cleavable capture oligonucleotides position thereon.
  • the cells and/or cell nuclei can be permeabilized for the delivery of oligonucleotide with spatial barcodes cleaved and released from the substrate.
  • the RNA transcripts and oligonucleotide with spatial barcodes within the same cells and/or cell nuclei can be captured by the single-cell barcoded capture probes being representative of the single-cell identity of the cells of the tissue sample.
  • the spatial information of cDNA of RNA transcripts with single-cell barcodes can be obtained from the reverse-transcribed oligonucleotides with spatial barcodes carrying same single-cell barcodes.
  • spatial and genomic information can be obtained from the barcoded cDNA of the polyadenylated RNA transcripts from the tissue sample, insofar as the location of the spatial barcodes on the substrate corresponds to the location of the cell (of the tissue sample) .
  • the method of DNA fragmentation, such as transposition, transcription and reverse transcription can be practiced in situ on cells of a tissue sample contacting microfluidic devices.
  • RNA transcripts can be polyadenylated, and the cells and/or cell nuclei can be permeabilized for the delivery of capture oligonucleotide probes from microfluidic devices.
  • Polyadenylated RNA can be captured and reverse transcribed by the delivered capture oligonucleotide probes being representative of the spatial positioning of the cells of the tissue sample.
  • spatial and genomic information can be obtained from the barcoded cDNA of the polyadenylated RNA from the tissue sample, insofar as the location of the barcodes on the substrate corresponds to the location of the cell (of the tissue sample) .
  • the method of DNA fragmentation such as transposition, transcription, and reverse transcription using random primer can be practiced in situ on cells of a tissue sample.
  • the tissue sample on the slide can be placed on a substrate having capture oligonucleotides position thereon.
  • the cells and/or cell nuclei can be permeabilized for the release of cDNA to be captured by the capture oligonucleotide probes with barcodes on the substrate thereby being representative of the spatial positioning of the cells of the tissue sample.
  • spatial barcoded can be added to the cDNA.
  • the disclosure provides a method combining a transposition system to make tagged fragments of genomic DNA, transcribing the tagged DNA fragments into RNA and polyadenylating the RNA to make RNA with poly (A) tails.
  • RNA can then be captured using a plurality of capture oligonucleotides with spatial barcodes and then reverse transcribed into cDNA for amplification/sequencing.
  • Exemplary capture oligonucleotides within the plurality may include a poly (T) portion, random primers or probes targeting specific sequences.
  • oligonucleotides can be used that include random primers for reverse transcription.
  • oligonucleotides with barcodes can be ligated to the post-reverse transcription cDNA, so as to add spatial barcodes.
  • DNA probes can be used that include target specific probes and a poly (A) tail to hybridize to RNA transcripts.
  • the poly (A) tails get captured by barcoded poly (T) capture oligonucleotides after cell permeabilized. DNA synthesis using the sequence specific probes is performed with barcoded poly (T) oligos as primers, so as to add spatial barcodes.
  • methods described herein integrating in situ DNA fragmentation, such as transposase-assisted tagmentation and transcription with polyadenylation provide spatially resolved genome-wide profiling of chromatin, such as open chromatin, which enables single-cell resolution.
  • the methods described herein integrating in situ DNA fragmentation such as transposase-assisted tagmentation and transcription with polyadenylation can be used with or without known sequencing-based spatial transcriptomic devices.
  • aspects of the present disclosure may be carried out on a single cell level or single DNA molecule level or with a plurality of cells.
  • the plurality of cells may be of the same cell type.
  • the plurality of cells may be of different cell type.
  • the plurality of cells may be within a tissue sample and DNA analysis of each cell is resolved on a single cell basis.
  • the plurality of cells may be within a tissue sample and DNA analysis of each cell is resolved on a single cell basis so as to provide spatial information of genomic DNA within the tissue sample.
  • the methods described herein integrating in situ DNA fragmentation such as transposase-assisted tagmentation and transcription with polyadenylation can be used with a single cell or a plurality of cells alone or in combination and need not be carried out on a tissue sample.
  • the methods described herein integrating in situ DNA fragmentation such as transposase-assisted tagmentation and transcription with polyadenylation can be carried out in situ on cells of a tissue sample as described herein when spatial genomic information
  • RNA transcripts are reverse transcribed in situ to first strand cDNA using random primers.
  • a poly (A) tail is then added in situ to the cDNA using methods known to those of skill in the art such as by using a template independent DNA polymerase.
  • Exemplary template independent polymerases are known to those of skill in the art and include terminal deoxynucleotidyl transferase ( “TdT” ) which adds a plurality of A nucleotides to the cDNA to form a poly (A) tail as is known in the art. See Yue et al., Curr. Protoc. Mol. Biol.
  • template-independent DNA polymerases include T4 and T7 ligases which adds a plurality of adaptors containing A nucleotides to the cDNA to form a poly (A) tail.
  • T4 and T7 ligases which adds a plurality of adaptors containing A nucleotides to the cDNA to form a poly (A) tail.
  • the cDNA with the poly (A) tail is then captured by barcoded poly (T) oligonucleotide capture probes after cell lysis as described herein.
  • Second strand synthesis is then carried out using poly (T) oligonucleotides as primers, thereby adding spatial barcodes.
  • RNA transcripts as described herein, sequence specific DNA probes including poly (A) tails are hybridized to the RNA transcripts.
  • the cDNA with the poly (A) tail is then captured by barcoded poly (T) oligonucleotide capture probes after cell lysis as described herein.
  • Second strand synthesis is then carried out using poly (T) oligonucleotides as primers, thereby adding spatial barcodes.
  • aspects of the present disclosure provides a method of analyzing genomic DNA of a cell comprising
  • step (e) further comprising performing second-strand synthesis by using the barcoded complementary DNA fragments as template to create products containing genomic information and with barcodes.
  • the method further comprising sequencing the plurality of complementary DNA fragments.
  • the method further comprising amplifying the plurality of complementary DNA fragments.
  • the method further comprising amplifying and sequencing the plurality of complementary DNA fragments.
  • steps (a) , (b) , and (c) are performed on a plurality of cells.
  • steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample.
  • steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample and the tissue sample is contacting a substrate comprising oligonucleotide capture probes.
  • step (c) is carried out by binding the plurality of RNA fragments to the oligonucleotide capture probes.
  • step (c) is carried out by binding the plurality of polyadenylated RNA fragments, which underwent in situ polyadenylation and carrying a poly (A) tail, to the oligonucleotide capture probes.
  • step (c) is carried out by the oligonucleotide capture probes delivered to a plurality of cells.
  • step (c) is carried out by the oligonucleotide capture probes delivered to a plurality of cells of a tissue sample.
  • step (c) is carried out by the oligonucleotide capture probes on the substrate.
  • step (c) is carried out by lysing the plurality of cells and the plurality of RNA fragments bind to the oligonucleotide capture probes on the substrate.
  • step (a) is carried out by a transposition system to create tagged DNA fragments.
  • step (a) is carried out by nuclease digestion to create DNA fragments.
  • the transposition system is a Tn5 transposition system or a MuA transposition system.
  • step (b) is carried out using a T7 RNA polymerase, T3 RNA polymerase, or Sp6 RNA polymerase.
  • the cell is a single cell and the plurality of isolated RNA fragments of step (c) corresponds to the single cell.
  • steps (a) and (b) are performed on a plurality of cells of a tissue sample and the plurality of RNA fragments of step (c) and (d) comprises RNA fragments corresponding to each of the plurality of cells.
  • the oligonucleotide capture probes comprise oligo (dT) sequences.
  • the oligonucleotide capture probes comprise sequences acting as random primer.
  • the oligonucleotide capture probes comprise sequences acting as sequence specific probes.
  • step (d) and (e) are using barcodes capable of indexing spatial positions.
  • step (d) and (e) are using barcodes capable of indexing single cells.
  • step (d) and (e) are using barcodes capable of indexing single cells and spatial positions.
  • step (d) and (e) are carried out through a direct barcoding after the reverse transcription with the oligonucleotide capture probes carrying barcoding information.
  • step (d) and (e) are carried out through a secondary barcoding based on a post-reverse transcription barcoding of complementary DNA fragments with oligonucleotide capture probes carrying barcoding information.
  • step (d) and (e) are carried out through a direct barcoding after the reverse transcription with the oligonucleotide capture probes carrying barcoding information and a post-reverse transcription barcoding of complementary DNA fragments with oligonucleotide capture probes carrying barcoding information.
  • the genomic DNA fragments comprise one or more open regions.
  • the genomic DNA is processed to enrich for open chromatin DNA.
  • the genomic DNA fragments comprise one or more targeted regions.
  • the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications.
  • the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications and transcription factor binding sites.
  • the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications, transcription factor binding sites and DNA modifications.
  • the genomic DNA fragments comprise one or more targeted regions enriched for cytosine converted to uracil by deamination.
  • the genomic DNA fragments comprise one or more targeted regions enriched for DNA methylation.
  • aspects of the present disclosure provides a method of analyzing genomic DNA of a cell comprising
  • Fig. 1 is a schematic depicting a method of spatial profiling of open chromatin.
  • Fig. 2 is a representative image of the transcripts inside nucleus after in situ tagmentation and in situ transcription as shown in Panel A and Panel B.
  • Fig. 3 is a schematic illustration of spatial profiling of histone modifications.
  • Fig. 4 is representative imaging of transcripts inside nucleus after in situ antibody directed tagmentation and in situ transcription.
  • Fig. 5 is representative imaging of the transcripts inside nucleus after in situ tagmentation (upper row) and in situ transcription (lower row) with different RNA polymerases.
  • Fig. 6 is representative imaging of the in situ transcripts inside nucleus after in situ tagmentation with Tn5 containing different transposon sequences (upper row and lower row) .
  • Fig. 7 depicts genome browser tracks of open-chromatin libraries from Hela cells.
  • Fig. 8 depicts data from experiments showing consistent performance across different open-chromatin profiling methods, as shown in Panel A, Panel B and Panel C.
  • Fig. 9 depicts data from experiments showing the efficiency of detecting unique fragments in single cells, as shown in Panel A and Panel B.
  • Fig. 10 depicts data from experiments of spatial barcoding of the cDNA of in situ transcripts with a spatial transcriptomic slide BMKMANU, as shown in Panel A and Panel B.
  • Fig. 11 depicts spatial distribution of chromatin accessibility in the hippocampal region of mouse brain on a BMKMANU S1000 Spatial Transcriptome Chip.
  • Fig. 12 is a schematic illustration of spatial barcoding of the cDNA of in situ transcripts with a spatial transcriptomic platform SeekSpace.
  • Fig. 13 depicts spatial distribution of chromatin accessibility in the mouse brain region using spatial transcriptomic platform SeekSpace.
  • Fig. 14 is a schematic illustration of spatial barcoding of the cDNA of in situ transcripts with a spatial transcriptomic platform Visium CytAssist.
  • Fig. 15 depicts spatial distribution of chromatin accessibility in the mouse brain region using spatial transcriptomic platform Visium CytAssist.
  • Fig. 16 depicts genome browser tracks of open-chromatin profiles detected by different methods from mouse brain sample.
  • Fig. 17 is a schematic illustration of spatial barcoding of the cDNA of in situ transcripts with a spatial microfuludic platform DBiT-seq.
  • Fig. 18 depicts data from experiments showing the efficiency of spatially detecting unique fragments in mouse embryo, as shown in Panel A, Panel B and Panel C.
  • Fig. 19 depicts data from experiments showing spatial distribution of chromatin activity of genes in the specific regions of mouse embryo.
  • Fig. 20 depicts data from experiments showing spatial distribution of transcription factor activity in the specific regions of mouse embryo.
  • Fig. 21 depicts data from experiments showing clustering of detected spots and their corresponding spatial distribution.
  • Fig. 22 depicts data from experiments showing aggregate fragments from spatially defined clusters in a representative genomic region.
  • aspects of the present disclosure are directed to methods for analyzing spatial genomic organization in complex tissues to obtain information about the functionality of diverse types of cells and unique gene expression based on local microenvironment and cellular coordination.
  • Methods described herein provide spatially resolved measurement of regulatory elements within chromatin structure.
  • Methods described herein utilize in situ DNA fragmentation such as transposase-assisted tagmentation, and in situ transcription.
  • methods described herein utilize in situ DNA fragmentation such as transposase-assisted tagmentation, and in situ transcription, and in situ polyadenylation.
  • the in situ DNA fragmentation, such as transposase-assisted tagmentation, and in situ transcription methods provide linearly amplified RNA fragments from genomic DNA.
  • the in situ polyadenylation method polyadenylates the RNA fragments for capture by poly (T) capture oligonucleotides.
  • the poly (A) tail of an RNA fragment can also be used to reverse transcribe the RNA into cDNA.
  • NGS next generation sequencing
  • Certain methods include microarray methods and microfluidic methods. Certain methods include barcoding of tissue sections.
  • tissue cryosections are created and the section is placed on a slide with barcoded capture oligonucleotides.
  • the section of tissue can be 10um in thickness which is around a layer of cells and is fixed by fixation reagents, such as glyoxal or paraformaldehyde (PFA) , on the slide.
  • fixation reagents such as glyoxal or paraformaldehyde (PFA)
  • Permeabilization of the tissue is carried out with reagents, such as pepsin in 0.01N HCl.
  • RNA with a polyA tail is hybridized to the capture oligonucleotide with a poly (T) sequence.
  • Reverse transcription is performed.
  • the first strand cDNA including the capture oligonucleotide with the poly (T) primer used during transcription contains RNA information and a spatial barcode.
  • the RNA is removed and, second strand synthesize is carried out using the cDNA first strand as a template to acquire spatial barcode.
  • the synthesized second strand with spatial barcode can be released and used for library preparation.
  • the RNA can be captured by a plurality of capture oligonucleotides containing random primers.
  • first strand cDNA including the capture oligonucleotide with the random primer used during transcription contains RNA information.
  • First strand cDNA synthesized by random primers are subjected to polyadenylation with terminal nucleotidyltransferase (TdT) .
  • First strand cDNA synthesized by random primers with polyA tails are captured by the capture oligonucleotide with a poly (T) sequence.
  • Second strand synthesis is performed and conferred a spatial barcode.
  • the second strand cDNA including the capture oligonucleotide with random primer used during transcription contains RNA information and a spatial barcode.
  • the RNA can be captured by a plurality of capture probes targeting specific sequences and containing polyA tail. Capture probes targeting specific sequences with polyA tails are further captured by the capture oligonucleotide with a poly (T) sequence. Second strand synthesis is performed and conferred a spatial barcode. The second strand product including the capture probes targeting specific sequences contains RNA information and a spatial barcode.
  • aspects of certain spatial transcriptomics devices contemplated by the present disclosure include a slide based on a microarray commercially available from Visium, a slide commercially available as Slide-seq, HDST (High Definition Spatial Transcriptomics) which uses a spatially barcoded bead array and BMKgene spatial gene expression slide based on oligo conjugated beads.
  • Systems commercially known as Stereo-seq, seq-scope and pixel-seq is based on a sequencing machine flowcell, using rolling cycle amplification or bridge amplification to generate oligo clusters.
  • the present disclosure also contemplates use of systems such as DBiT-Seq which is a microfluidic-based method which delivers barcodes to the surface of a tissue slide to allow for spatial omics sequencing. Accordingly, the present disclosure contemplates the use of spatial transcriptomics devices including slides having barcoded microarrays thereon, slides having barcoded beads thereon, or microfluidic systems for spatially barcoding polyadenylated RNA on tissue as described herein.
  • Methods of the present disclosure combine DNA fragmentation methods, such as transposition methods, with transcription methods in an in situ setting for genomic and epigenetic analyses in single cells and spatial biology.
  • T7 promoter and Tn5 transposase mosaic end (ME) sequence are used.
  • Tn5 tagmentation is carried out inside the nucleus where the genomic DNA is fragmented and tagged by transposon with T7 promoter sequence.
  • Alternate DNA fragmentation methods can be used, such as nuclease fragmentation followed by ligation of a transcriptase promoter sequence. Following tagmentation, gap-filling is performed to generate a double-stranded T7 promoter.
  • T7 RNA polymerase is then used for transcription within the nucleus such as by using materials and methods found in in vitro transcription (IVT) kits, to amplify the genomic DNA fragments into genomic RNAs.
  • IVT in vitro transcription
  • the genomic RNAs are then subject to enzymatic in situ polyadenylation of the T7 transcripts inside nucleus.
  • poly (A) polymerase mediated polyadenylation the T7 transcripts mimic the structure of natural mRNA with poly (A) tail, which can be reverse-transcribed into cDNA for library preparation using oligo (dT) primer.
  • a section of mouse brain is attached on a commercial spatial gene expression slide (BMKMANU S1000 Spatial Transcriptome Chip) from BMKGENE, whereby high-quality chromatin accessibility data containing positional information from the mouse brain was produced as described herein.
  • the BMKManu S1000 Spatial Transcriptome Chip has an enhanced resolution of 5 ⁇ M, reaching the subcellular range, and enabling multi-level resolution settings.
  • the S1000 chip includes approximately 2 million spots, and uses microwells layered with beads loaded with spatially barcoded capture probes.
  • the embedded mouse brain was cut into a thickness of 10 um (around one layer of cells) and placed on a capture chip with unique positional barcodes.
  • tissue sections on the chip were permeabilized, allowing T7 transcripts to be captured by the poly (T) oligonucleotides on the chip.
  • T7 transcripts After reverse transcription, RNA removal and second-strand synthesis, the resulting second-strand cDNAs were amplified to produce a library for sequencing.
  • genomic analysis methods described herein utilizing exemplary transposition, transcription and polyadenylation may or may not utilize a spatial gene expression slide.
  • Genomic analysis methods described herein utilizing exemplary transposition, transcription and polyadenylation may or may not utilize other spatial gene expression materials and methods known to those of skill in the art.
  • in situ DNA fragmentation such as transposition, transcription and polyadenylation, methods described herein produce numerous copies of polyadenylated RNAs.
  • polyadenylated RNAs can be directly captured by poly (T) capture oligonucleotides, such as those positioned on a spatial transcriptome chip, using standard methods and workflows, insofar as the poly (A) tail binds to the poly (T) capture oligonucleotides.
  • the captured RNA can then be reverse transcribed into cDNA producing barcoded cDNA, thereby avoiding prior art approaches that ligate DNA fragments to barcoded capture oligonucleotides.
  • the in situ DNA fragmentation such as tagmentation, transcription and polyadenylation described herein collectively enhance the sensitivity of spatial genomic profiling.
  • the genomic DNA is fragmented and tagged by transposons with a T7 promoter sequence. Following tagmentation, gap-filling is performed to generate a double-stranded T7 promoter. Then, T7 RNA polymerase is used for in situ transcription to amplify the genomic DNA fragments into genomic RNAs. Enzymatic in situ polyadenylation of the transcripts produced by the T7 RNA polymerase is carried out within the nucleus.
  • the transposon includes a T7 promoter and Tn5 transposase mosaic end (ME) sequence. Therefore, the starting region of all T7 transcripts include an ME sequence.
  • a fluorescent oligo that is complementary to the ME sequence of the transcripts is used as a label for the transcripts within the nucleus.
  • the T7 RNA polymerase can be replaced by an Sp6 or a T3 RNA polymerase or a similar RNA polymerase by changing the promoter sequence in the transposon.
  • flanking sequences for the T7 promoter sequence, and other similar promoter sequences can be optimized, and may be linear oligonucleotide sequences, as opposed to having a looping structure, as is known in the art.
  • Cells according to the invention include any cell where understanding single cell genomics or spatial genomics of a tissue sample is considered by those of skill in the art to be useful.
  • Cells include prokaryotic cells or eukaryotic cells.
  • a cell according to the present disclosure includes a cancer cell of any type, hepatocyte, oocyte, embryo, stem cell, iPS cell, ES cell, neuron, erythrocyte, melanocyte, astrocyte, germ cell, oligodendrocyte, kidney cell and the like.
  • Cells useful in the methods described herein can be obtained from a biological sample, tissue of interest, or from a biopsy, blood sample, or cell culture. Additionally, cells from specific organs, tissues, tumors, neoplasms, or the like can be obtained and used in the methods described herein. Furthermore, in general, cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic single celled organisms including bacteria or yeast. According to one aspect, the sample may be in vitro.
  • the term “in vitro” has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts.
  • biological sample is intended to include, but is not limited to, tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the methods of the present invention are practiced with a single cell or a plurality of cells with single cell resolution.
  • a single cell refers to one cell.
  • a single cell suspension can be obtained using standard methods known in the art including, for example, enzymatically using trypsin or papain to digest proteins connecting cells in tissue samples or releasing adherent cells in culture, or mechanically separating cells in a sample.
  • Single cells can be placed in any suitable reaction vessel in which single cells can be treated individually. For example, a 96-well plate, such that each single cell is placed in a single well.
  • Methods for manipulating single cells include fluorescence activated cell sorting (FACS) , flow cytometry (Herzenberg., PNAS USA 76: 1453-55 1979) , micromanipulation and the use of semi-automated cell pickers (e.g. the QUIXELL cell transfer system from Stoelting Co. ) .
  • Individual cells can, for example, be individually selected based on features detectable by microscopic observation, such as location, morphology, or reporter gene expression.
  • a combination of gradient centrifugation and flow cytometry can also be used to increase isolation or sorting efficiency.
  • the methods of the present invention are practiced with a plurality of cells with single cell resolution.
  • a plurality of cells includes from about 2 to about 1,000,000 cells, about 2 to about 10 cells, about 2 to about 100 cells, about 2 to about 1,000 cells, about 2 to about 10,000 cells, about 2 to about 100,000 cells, about 2 to about 10 cells or about 2 to about 5 cells.
  • the methods of the present invention are practiced with a tissue sample, where a section of tissue is created, with an exemplary single layer of cells as described herein. Utilizing a tissue sample provides spatial genomic information of the cells making up the tissue sample.
  • the DNA to be treated is genomic DNA or chromatin DNA.
  • the DNA to be treated is mammalian DNA, plant DNA, yeast DNA, viral DNA, or prokaryotic DNA.
  • the DNA sample is obtained from a human, bovine, porcine, ovine, equine, rodent, avian, fish, shrimp, plant, yeast, virus, or bacteria.
  • the DNA to be treated is genomic DNA.
  • the term "genome” as used herein is defined as the collective gene set carried by an individual, cell, or organelle.
  • genomic DNA as used herein is defined as DNA material comprising the partial or full collective gene set carried by an individual, cell, or organelle.
  • the DNA to be treated is a double stranded polynucleotide molecule with protein binding thereto, such as cell-free DNA.
  • DNA such as genomic DNA or chromatin DNA
  • genomic DNA may be isolated and treated with a transposition system, a transcriptase and a polyadenylation enzyme as described herein.
  • the genomic DNA is in situ within a cell nucleus.
  • the cell or cell or nucleus or nuclei are treated with a crosslinking agent to maintain cellular structure as is known in the art before treatment with DNA fragmentation agents, transcription agents or polyadenylation agents.
  • a crosslinking agent to maintain cellular structure as is known in the art before treatment with DNA fragmentation agents, transcription agents or polyadenylation agents.
  • Such crosslinking treatments include treatment with glyoxal or PFA.
  • Such crosslinking treatments include treatment with methanol.
  • Such crosslinking treatments include treatment with paraformaldehyde or treatment with ultra violet light to effect crosslinking.
  • the crosslinking is used to maintain cellular structure.
  • Methods described herein can be applied to cells and nuclei treated with a crosslinking agent. According to one aspect, methods described herein can be applied to Formalin-Fixed and Paraffin-Embedded (FFPE) samples and the like, as is known in the art.
  • FFPE Formalin-Fixed and Paraffin-Embedded
  • FFPE is a form of preservation and preparation of specimens.
  • a tissue sample is first preserved by fixing it in formaldehyde, also known as formalin, such as a solution of 10%neutral-buffered formalin for about 18-24 hours, to preserve the proteins and vital structures within the tissue.
  • formalin such as a solution of 10%neutral-buffered formalin for about 18-24 hours
  • the tissue is embedded in a paraffin wax block, samples of which can then be processed according to the methods described herein.
  • the tissue may be dehydrated and cleared, often using increasing concentrates of ethanol. Then, it is embedded into IHC-grade paraffin according to known methods.
  • the methods described herein may be practiced on the cell or cells or a nucleus or nuclei of the cell or cells.
  • An individual cell or plurality of cells or nucleus or nuclei may be isolated or may be within a tissue section.
  • the cell or cells or nucleus or nuclei may be treated according to known methods to facilitate entry of a transposition system, a translation system and a polyadenylation system or other reagents to be introduced into the cell or cells or nucleus or nuclei.
  • the cell or cells or a nucleus or nuclei may be permeabilized.
  • Methods of permeabilization are known to those of skill in the art. Exemplary permeabilization techniques include electroporation or electropermeabilization, permeabilization with 0.1N or 0.01N HCl, permeabilization with mild non-ionic detergents such as Tween-20, IGEPAL CA-630, TritonX-100, saponin and digitonin and by pore-forming toxins, such as alpha-toxin and streptolysin O, as is known in the art.
  • Electroporation is a technique in which an electrical field is applied to cells or the nuclei of cells in order to increase the permeability of the cell membrane, allowing a transposition system, a translation system and a polyadenylation system or other reagents to be introduced into the cell.
  • the cell or cells may be lysed to obtain the nucleus or nuclei, or the nucleus or nuclei may be lysed, using methods known to those of skill in the art. Lysis can be achieved by, for example, heating the cells, or by the use of detergents or other chemical methods, or by a combination of these. However, any suitable lysis method known in the art can be used.
  • DNA such as genomic DNA or chromatin DNA
  • DNA extraction protocols using beads such as DYNABEADS
  • reagents are known to those of skill and are commercially available in kits through ThermoFisher Scientific, for example CHARGESWITCH genomic DNA purification kits, and the like.
  • chromatin DNA is processed using a transposition method which may be referred to in the art as transposome mediated fragmentation or “tagmentation” .
  • transposome mediated fragmentation or “tagmentation” .
  • tagmentation transposomes are prepared with DNA that is afterwards cut so that the transposition events result in fragmented DNA with adapters.
  • target DNA is simultaneously fragmented and tagged producing fragments tagged with desired DNA sequences for downstream processing.
  • Tagmentation methods and reagents in general are disclosed in US20110287435. For additional useful methods to make sequencing libraries, see Single-cell chromatin accessibility reveals principles of regulatory variation.
  • an exemplary transposon system includes Tn5 transposase, Mu transposase, Tn7 transposase or IS5 transposase and the like.
  • Other useful transposon systems are known to those of skill in the art and include Tn3 transposon system (see Maekawa, T., Yanagihara, K., and Ohtsubo, E. (1996) , A cell-free system of Tn3 transposition and transposition immunity, Genes Cells 1, 1007-1016) , Tn7 transposon system (see Craig, N.L. (1991) , Tn7: a target site-specific transposon, Mol. Microbiol.
  • Tn10 tranposon system see Chalmers, R., Sewitz, S., Lipkow, K., and Crellin, P. (2000) , Complete nucleotide sequence of Tn10, J. Bacteriol 182, 2970-2972
  • Piggybac transposon system see Li, X., Burnight, E.R., Cooney, A.L., Malani, N., Brady, T., Sander, J.D., Staber, J., Wheelan, S.J., Joung, J.K., McCray, P.B., Jr., et al. (2013) , PiggyBac transposase tools for genome engineering, Proc. Natl. Acad. Sci.
  • the transposition system is a MuA transposition system.
  • treated genomic DNA is contacted with Tn5 transposases each bound to a transposon DNA, to form a transposase/transposon DNA complex dimer called a transposome.
  • the transposome bind to target locations along the treated genomic DNA and cleave the treated genomic DNA into a plurality of double stranded fragments with primer binding sites. Processing, such as extension and gap filling may take place to produce a double stranded product which may optionally be transcribed by RNA polymerase, or mixed with primers together with a DNA polymerase, nucleotides and amplification reagents, and the double stranded genomic DNA fragment is amplified.
  • the amplicons are sequenced using, for example, high-throughput sequencing methods known to those of skill in the art. See WO2017/015075.
  • a targeted chromatin region such as an open chromatin region, may be treated using a nuclease to create DNA fragments within the nucleus which are then ligated to a transcriptase promoter, such as is used with an RNA polymerase.
  • Nucleases are an alternate embodiment to using a transposition system to create DNA fragments, where the fragments are ligated to a transcriptase promoter.
  • a nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. There are two primary classifications based on the locus of activity.
  • Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules. According to certain aspects, a nuclease may be used to process DNA into fragments before the fragments are transcribed into RNA. Such fragments may then be transcribed into RNA and polyadenylated as described herein. Exemplary nucleases include DNase (such as DNase I commercially available from Thermo Fisher) , MNase (micrococcal nuclease commercially available from New England Biolabs) or a restriction endonuclease (such as FASTDIGEST commercially available from Thermo Fisher) and the like.
  • DNase such as DNase I commercially available from Thermo Fisher
  • MNase micrococcal nuclease commercially available from New England Biolabs
  • FASTDIGEST commercially available from Thermo Fisher
  • the genomic DNA may be pre-processed in a manner to convert cytosines to uracils as is known in the art or to fragment the genomic DNA to enrich for particular locations of the genomic DNA before the in situ tagmentation, in situ transcription and in situ polyadenylation described herein is carried out.
  • cytosines to uracils as is known in the art
  • fragment the genomic DNA to enrich for particular locations of the genomic DNA before the in situ tagmentation, in situ transcription and in situ polyadenylation described herein is carried out.
  • Tn5 transposase when Tn5 transposase is added to cell of naive state, resulting genomic fragments are enriched in open regions.
  • antibodies targeting histone modifications, DNA modifications, or transcription factors
  • Pre-treating chromatin with HCL to remove nucleosomes before adding Tn5 ensures uniform genome fragmentation across the whole genome.
  • the present disclosure is directed to adding spatial barcodes to genomic fragments within cells.
  • the genomic fragments may be created by methods other than transposition systems to enrich for certain genomic DNA as desired before the in situ tagmentation, in situ transcription and in situ polyadenylation described herein is carried out. Additionally, the genomic DNA may be treated to alter the genomic DNA before fragmentation, such as methylation detection experiments using dsDNA deaminase or determining transcription factor binding sites, resulting from base conversion. Even after base conversion and optional fragmentation using methods other than a transposition system, the fragmented genome can still be utilized in the in situ tagmentation, in situ transcription and in situ polyadenylation method described herein.
  • the in situ tagmentation, in situ transcription and in situ polyadenylation method described herein is compatible with many known genomic analysis techniques that utilize the addition of spatial barcodes to mRNA including poly (A) tails, such as spatial barcode slides or microfluidics techniques.
  • a targeted chromatin region such as an open chromatin region
  • a targeted chromatin region may be enriched using methods known to those of skill in the art including ATAC, ChIC, ChEC, ChEC-seq, CUT&TAG, CUT&RUN, Multi-CUT&Tag, NTT-seq, R loop CUT&Tag and the like, in addition to methods that use a transposase, such as Tn5 transposase.
  • CUT&Tag-sequencing also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA.
  • CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. See "CUT&Tag: a higher resolution, lower cost way to map chromatin" . Fred Hutchinson Cancer Research Center. 29 April 2019.
  • CUT&RUN sequencing (see US 2022/0214356) , also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA.
  • CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. See “Lay off the ChIPs: CUT&RUN instead” . Fred Hutchinson Cancer Research Center. 20 February 2017.
  • ChIC detects the binding sites of transcription factors in the genome by targeting a modified micrococcal nuclease (MNase) , conjugated with protein A (pA-MN) , using a specific antibody.
  • MNase micrococcal nuclease
  • pA-MN protein A
  • the modified MNase specifically cleaves DNA at regions interacting with a protein of interest only when Ca2+ ions are present, therefore allowing for controlled DNA cleavage at the antibody binding site.
  • This approach allows for mapping proteins with a 100–200 bp resolution and excellent specificity. See Schmid M, Durussel T, Laemmli UK. 2004. ChIC and ChEC. Molecular Cell. 16 (1) : 147-15.
  • ChEC-seq Chromatin endogenous cleavage
  • MNase micrococcal nuclease
  • ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. See Grunberg et al., J. Vis. Exp. 2017; (124) e55836, p. 1-9.
  • genomic DNA within the cell nuclease is subjected to ChIP-seq, ChIC, ChEC, ChEC-seq, CUT&TAG, CUT&RUN, Multi-CUT&Tag, NTT-seq, R loop CUT&Tag processing in order to enrich chromatin regions.
  • exemplary target DNA sequences are open chromatin, which can be targeted by ATAC-seq, for example.
  • method described herein have application to detect histone modifications in a spatial context.
  • CUT&TAG is used to map transcription factors.
  • exemplary target DNA sequences include fragments of the entire genome.
  • Slide-DNA-seq as described in Spatial genomics enables multi-modal study of clonal heterogeneity in tissues.
  • Nature 601.7891 (2022) : 85-91 captures fragments from the entire genome.
  • the genomic DNA is pretreated with 0.1N HCl before tagmentation. This treatment removes histones from DNA while preserving the nucleus.
  • Tn5 tagmentation after HCl pretreatment enables fragment capture at any genomic position, not limited to open chromatin. Accordingly, methods described herein can be used with whole genome amplification.
  • exemplary target DNA sequences include fragments of the genome with cytosine to uracil conversion.
  • Certain DNA methylation detection methods rely on the conversion of cytidines on dsDNA, as evidenced in Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nature biotechnology 37.4 (2019) : 424-429 and Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.
  • Molecular Cell (2024) transcription factor binding sites can be identified by conversion from cytosine to uracil on dsDNA. See WO 2024/065721 hereby incorporated by reference in its entirety.
  • T7 RNA polymerase is known to capable of amplifying templates containing uracil template, such as RNA template. Accordingly, methods described herein can be used with amplification of genome with cytosine to uracil conversion.
  • the methods disclosed herein comprise transcription of a DNA construct as described herein to RNA whether in situ or in vitro. Transcription includes use of a linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase.
  • a transposition system to create RNA fragments from the DNA fragments created by the transposition system. See WO2017/015075 hereby incorporated by reference in its entirety for the teaching of transcription materials and methods.
  • An exemplary commercially available transcription kit includes HISCRIBE TM T7 Quick High Yield RNA Synthesis Kit commercially available from New England Biolabs.
  • the HISCRIBE TM T7 Quick High Yield RNA Synthesis Kit is designed for quick set-up and production of large amounts of RNA in vitro.
  • the reaction can be set up conveniently by combining the NTP buffer mix, T7 RNA Polymerase mix and a suitable DNA template.
  • the kit also allows for capped RNA or dye-labeled RNA synthesis by incorporation of cap analog (ARCA) or dye-modified nucleotides.
  • a DNA template such as linearized plasmid DNA, PCR products or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with the HISCRIBE TM T7 Quick High Yield RNA Synthesis Kit, provided that the DNA template contains a double-stranded T7 promoter region upstream of the sequence to be transcribed.
  • a minimal T7 promoter sequence is known in the art.
  • Components of commercially available transcription kits include DNase I, T7 RNA polymerase mix, and associated solutions and buffers.
  • the methods disclosed herein comprise amplification of nucleic acids including, for example, polynucleotides, oligonucleotides and/or oligonucleotide fragments, such as the barcodes described herein.
  • Amplification methods may comprise contacting a nucleic acid sequence with one or more primers (e.g., primers that are complementary to barcode sequences or sequences flanking the barcodes, such as for purposes of obtaining sequencing libraries) that specifically hybridize to the nucleic acid under conditions that facilitate hybridization and chain extension.
  • primers e.g., primers that are complementary to barcode sequences or sequences flanking the barcodes, such as for purposes of obtaining sequencing libraries
  • Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant.
  • isothermal amplification e.g., isothermal bridge amplification (IBA) , rolling circle amplification (RCA) , hyperbranched rolling circle amplification (HRCA) , strand displacement amplification (SDA) , helicase-dependent amplification (HDA) , PWGA or any other nucleic acid amplification method using techniques well known to those of skill in the art.
  • IBA isothermal bridge amplification
  • RCA rolling circle amplification
  • HRCA hyperbranched rolling circle amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent amplification
  • PWGA any other nucleic acid amplification method using techniques well known to those of skill in the art.
  • PCR Polymerase chain reaction, or “PCR, ” refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA.
  • PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
  • the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
  • a double stranded target nucleic acid may be denatured at a temperature greater than 90 °C, primers annealed at a temperature in the range 50-75 °C, and primers extended at a temperature in the range 72-78 °C.
  • a double stranded target nucleic acid may be denatured at a temperature greater than 90 °C in a conventional PCR using Taq DNA polymerase, or by adding formamide at 60 °C in isothermal bridge amplification using Bst polymerase.
  • PCR encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 microliters.
  • Reverse transcription PCR, ” or “RT-PCR, ” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al., U.S. Patent No. 5,168,038.
  • Real-time PCR means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds.
  • Nested PCR means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon.
  • initial primers in reference to a nested amplification reaction mean the primers used to generate a first amplicon
  • secondary primers mean the one or more primers used to generate a second, or nested, amplicon.
  • Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al. (1999) Anal. Biochem., 273: 221-228 (two-color real-time PCR) . Usually, distinct sets of primers are employed for each sequence being amplified. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen.
  • the amplicons are sequenced using, for example, high-throughput sequencing methods known to those of skill in the art. Determination of the sequence of a nucleic acid sequence of interest can be performed using a variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH) , sequencing by ligation (SBL) (Shendure et al. (2005) Science 309: 1728) , quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS) , stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET) , molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ) , FISSEQ beads (U.S. Pat. No.
  • SBH sequencing by hybridization
  • SBL sequencing by ligation
  • QIFNAS quantitative incremental fluorescent nucleotide addition sequencing
  • FRET fluorescence resonance energy transfer
  • molecular beacons TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing
  • allele-specific oligo ligation assays e.g., oligo ligation assay (OLA) , single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout
  • OLA oligo ligation assay
  • RCA rolling circle amplification
  • RCA rolling circle amplification
  • RCA rolling circle amplification
  • High-throughput sequencing methods e.g., using platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms, Ion Torrent semiconductor sequencing technology, single-molecule real-time (SMRT) sequencing from Pacific Biosciences, Nanopore-based sequencing from Oxford Nanopore Technologies, and the like, can also be utilized.
  • platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms, Ion Torrent semiconductor sequencing technology, single-molecule real-time (SMRT) sequencing from Pacific Biosciences, Nanopore-based sequencing from Oxford Nanopore Technologies, and the like.
  • the amplified DNA can be sequenced by any suitable method.
  • the amplified DNA can be sequenced using a high-throughput screening method, such as Applied Biosystems’ SOLiD sequencing technology, or Illumina's Genome Analyzer.
  • the amplified DNA can be shotgun sequenced.
  • the number of reads can be at least 10,000, at least 1 million, at least 10 million, at least 100 million, or at least 1000 million.
  • the number of reads can be from 10,000 to 100,000, or alternatively from 100,000 to 1 million, or alternatively from 1 million to 10 million, or alternatively from 10 million to 100 million, or alternatively from 100 million to 1000 million.
  • a "read” is a length of continuous nucleic acid sequence obtained by a sequencing reaction.
  • “Shotgun sequencing” refers to a method used to sequence very large amount of DNA (such as the entire genome) .
  • the DNA to be sequenced is first shredded into smaller fragments which can be sequenced individually.
  • the sequences of these fragments are then reassembled into their original order based on their overlapping sequences, thus yielding a complete sequence.
  • “Shredding" of the DNA can be done using a number of difference techniques including restriction enzyme digestion or mechanical shearing. Overlapping sequences are typically aligned by a computer suitably programmed. Methods and programs for shotgun sequencing a DNA library are well known in the art.
  • Particularly exemplary sequencing methods include Sanger sequencing (AB 13730x1 genome analyzer ) , pyrosequencing on a solid support (454 sequencing, Roche) , sequencing by-synthesis with reversible terminations (ILLUMINA Genome Analyzer) , DNA nanoball sequencing (DNBSEQ, MGI) , sequencing-by-ligation (ABI SOLID) or sequencing-by-synthesis with virtual terminators (HELI ) .
  • Other next generation sequencing techniques for use with the disclosed methods include Massively parallel signature sequencing (MPSS) , Polony sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing, Pacbio sequencing and Nanopore DNA sequencing.
  • Embodiments of the present disclosure employ spatial barcoding as is known in the art.
  • a capture spatial barcoded slide is provided including poly (T) oligonucleotide capture probes barcoded so that polyadenylated RNA bound to the poly (T) oligonucleotide capture probes provide spatial information.
  • Deterministic Barcoding in Tissue as described in High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue.
  • Cell 183.6 (2020) : 1665-1681 may be used to provide spatial information.
  • DBiT employs microfluidics to load barcodes into tissues.
  • DBiT is used to append barcodes to T7 transcripts.
  • Slide-tag as described in 'Slide-tags Enables Single-Nucleus Barcoding for Multimodal Spatial Genomics' is used. Slide-tag utilizes a spatial barcoded slide differently. Rather than capturing oligonucleotides from tissue, Slide-tag releases barcoded oligonucleotides into cells.
  • Embodiment 1 A method of analyzing genomic DNA of a cell comprising
  • Embodiment 2 The method of Embodiment 1 further comprising sequencing the plurality of complementary DNA fragments.
  • Embodiment 3 The method of Embodiment 1 further comprising amplifying the plurality of complementary DNA fragments.
  • Embodiment 4 The method of Embodiment 1 further comprising amplifying and sequencing the plurality of complementary DNA fragments.
  • Embodiment 5 The method of Embodiment 1 wherein steps (a) , (b) , and (c) are performed on a plurality of cells.
  • Embodiment 6 The method of Embodiment 1 wherein steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample.
  • Embodiment 7 The method of Embodiment 1 wherein steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample and the tissue sample is contacting a substrate comprising oligonucleotide capture probes.
  • Embodiment 8 The method of Embodiment 7 wherein step (d) is carried out by lysing the plurality of cells and the plurality of polyadenylated RNA fragments bind to the oligonucleotide capture probes on the substrate.
  • Embodiment 9 The method of Embodiment 1 wherein step (a) is carried out by a transposition system to create tagged DNA fragments.
  • Embodiment 10 The method of Embodiment 1 wherein step (a) is carried out by nuclease digestion to create DNA fragments.
  • Embodiment 11 The method of Embodiment 9 wherein the transposition system is a Tn5 transposition system or a MuA transposition system.
  • Embodiment 12 The method of Embodiment 1 wherein step (b) is carried out using a T7 RNA polymerase, T3 RNA polymerase, or Sp6 RNA polymerase.
  • Embodiment 13 The method of Embodiment 1 wherein the cell is a single cell and the plurality of isolated polyadenylated RNA fragments of step (d) corresponds to the single cell.
  • Embodiment 14 The method of Embodiment 7 wherein steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample and the plurality of isolated polyadenylated RNA fragments of step (d) comprises polyadenylated RNA fragments corresponding to each of the plurality of cells.
  • Embodiment 15 The method of Embodiment 7 wherein the oligonucleotide capture probes comprise oligo (dT) sequences.
  • Embodiment 16 The method of Embodiment 7 wherein the oligonucleotide capture probes comprise sequences acting as random primer.
  • Embodiment 17 The method of Embodiment 7 wherein the oligonucleotide capture probes comprise sequences acting as sequence specific probes.
  • Embodiment 18 The method of Embodiment 1 wherein step (e) and (f) are using barcodes capable of indexing spatial positions.
  • Embodiment 19 The method of Embodiment 1 wherein step (e) and (f) are using barcodes capable of indexing single cells.
  • Embodiment 20 The method of Embodiment 1 wherein step (e) and (f) are using barcodes capable of indexing single cells and spatial positions.
  • Embodiment 21 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more open regions.
  • Embodiment 22 The method of Embodiment 1 wherein the genomic DNA is processed to enrich for open chromatin DNA.
  • Embodiment 23 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions.
  • Embodiment 24 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications.
  • Embodiment 25 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications and transcription factor binding sites.
  • Embodiment 26 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions enriched for certain histone modifications, transcription factor binding sites and DNA modifications.
  • Embodiment 27 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions enriched for cytosine converted to uracil by deamination.
  • Embodiment 28 The method of Embodiment 1 wherein the genomic DNA fragments comprise one or more targeted regions enriched for DNA methylation.
  • Embodiment 29 A method of analyzing genomic DNA of a cell comprising
  • Oligonucleotides for Tn5 and pAG-Tn5 transposome adaptor were synthesized at Sangon and are shown in Table 1 below. (SEQ ID NOS: 1-13) (N is A, T, C, or G, B is T, C, or G and V is A, C, or G)
  • oligo mixtures were denatured for 5 min at 95 °C and cooled down slowly (-0.1 °C/sto 16 °C) for annealing using a thermocycler.
  • ATAC-seq as described in Buenrostro, Jason D., et al. "Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.
  • the Tn5 transposome was assembled with the following components: 3.5 ⁇ l annealed double-stranded oligo S5-ME/ME-Rev, 3.5 ⁇ l annealed double-stranded oligo S7-ME/ME-Rev, 10 ⁇ l Tn5 Transposase (2 ⁇ g/ ⁇ l, cat. no. S111, Vazyme) and 33 ⁇ l Coupling buffer (supplied in kit) .
  • the Tn5 transposome was assembled with the following components: 7 ⁇ l annealed double-stranded oligo (containing RNA polymerase promoter sequence) , 10 ⁇ l Tn5 Transposase (2 ⁇ g/ ⁇ l, cat. no. S111, Vazyme) and 33 ⁇ l Coupling buffer (supplied in kit) .
  • the pAG-Tn5 transposome was assembled with the following components: 7 ⁇ l annealed double-stranded oligo (containing RNA polymerase promoter sequence) , 40 ⁇ l pAG-Tn5 Transposase (500 ng/ ⁇ l, cat. no.
  • HeLa cells were grown on BeyoGold TM 35mm Confocal Dishes (cat. no. FCFC016, Beyotime) using RPMI 1640 medium (cat. no. 11875093, Gibco) containing 10%FBS (cat. no. 10099141C, Gibco) in the 5%CO 2 incubator at 37°C until 80–90%confluent.
  • RPMI 1640 medium cat. no. 11875093, Gibco
  • 10%FBS cat. no. 10099141C, Gibco
  • a standard trypsinization was performed with 0.25% (w/v) Trypsin solution (cat. no. 15050065, Thermofisher) , and the trypsinized cells were resuspended in DPBS (cat. no. 14040117, Thermofisher) -0.5%BSA (cat. no.
  • the OCT embedded tissues were cut into a thickness of 10mm (around one layer of cells) using a Leica CM1950 cryostat, then placed on Superfrost Plus TM Slide (cat. no. 48311-703, VWR) or commercial spatial gene expression slide (BMKMANU S1000 Spatial Transcriptome Chip) , and stored at -80 °C until ready to use.
  • HeLa cells growing on confocal dishes or tissue sections were crosslinked in 0.2 ml of freshly prepared glyoxal fixation buffer (3%glyoxal, 0.75%glacial acetic acid, pH 5.0) at room temperature (RT) for 7 min.
  • the fixation buffer can be replaced by PFA (0.1%-4%percentage in PBS) .
  • PFA 0.1%-4%percentage in PBS
  • the cells were rinsed twice with 0.5 ml of DPBS (cat. no. 14040117, Thermofisher) -0.5%BSA (cat. no. A1933, Sigma) .
  • Standard ATAC-seq was performed using dissociated fresh cells. A total of 50,000 HeLa cells were centrifuged at 500 ⁇ g for 5 min, which was followed by a permeabilization using ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1%IGEPAL CA-630) for 5 min on ice. After permeabilization, nuclei were centrifuged at 500 ⁇ g for 5 min, and resuspended with 50 ⁇ l tagmentation mixture containing 33 mM Tris-acetate (pH 8) , 66 mM potassium acetate, 10 mM magnesium acetate, 16%dimethylformamide (cat. no.
  • the adherent cells (either HeLa cells growing on confocal dishes or tissue sectioned on slide) were permeabilized with 100 ⁇ l lysis buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1%IGEPAL CA-630) for 5 min on ice, and the supernatant was removed. Following permeabilization, cells were blocked with 120 ⁇ l DPBS (cat. no. 14040117, Thermofisher) -2.5%BSA (cat. no. A1933, Sigma) at RT for 1h.
  • DPBS cat. no. 14040117, Thermofisher
  • BSA cat. no. A1933, Sigma
  • a 1 100 dilution of pAG-Tn5 transposome was added into 120 ⁇ l Dig-300 Buffer, and was incubated with the cells at RT for 45 min.
  • the high salt Dig-300 Buffer prevents non-specific binding of pAG-Tn5 to genomic DNA.
  • Cells were rinsed twice with 150 ⁇ l Dig-300 Buffer to remove unbound pAG-Tn5.
  • 150 ⁇ l Dig-300 Buffer containing 10 mM MgCl2 were added to the cell and incubated in humid chamber box at 37°C for 45 min to activate transposition. After washing with 150 ⁇ l DPBS (cat. no.
  • the gap-filling reaction was performed by adding 120 ⁇ l 1X NEBuffer 2 (cat. no. B7002S, NEB) containing 3.3 ⁇ l of dNTP mix (cat. no. N0447, NEB) , 1.2 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) and 2 ⁇ l Klenow 3’ ⁇ 5’ exo- (cat. no. M0212, NEB) for 10 min at 25°C. Directly following transposition, the sample was placed on ice to stop the reaction.
  • adherent cells either HeLa cells growing on confocal dishes or tissue sectioned on slide
  • 100 ⁇ l lysis buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1%IGEPAL CA-630) for 5 min on ice.
  • the cells were rinsed once with 150 ⁇ l DPBS (cat. no. 14040117, Thermofisher) and the supernatant was removed.
  • the gap-filling reaction was performed by adding 120 ⁇ l 1X NEBuffer 2 (cat. no. B7002S, NEB) containing 3.3 ⁇ l of dNTP mix (cat. no. N0447, NEB) , 1.2 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) and 2 ⁇ l Klenow 3’ ⁇ 5’ exo- (cat. no. M0212, NEB) for 10 min at 25°C. Directly following transposition, the sample was placed on ice to stop the reaction.
  • cells were covered by a mixture of following components: 69.6 ⁇ l H2O, 12 ⁇ l 10X RNAPol Reaction Buffer (cat. no. B9012S, NEB) , 12 ⁇ l 0.1M DTT (cat. no. 707265ML, Thermofisher) , 2.4 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) , 1.2 ⁇ l SUPERase ⁇ In TM RNase Inhibitor (cat. no. AM2694, Thermofisher) , 1.2 ⁇ l Protector RNase Inhibitor (cat. no.
  • in situ polyadenylation was performed by adding 120 ⁇ l PAP enzyme mix containing 85.2 ⁇ l H2O, 6 ⁇ l E. coli Poly (A) Polymerase (cat. no. M0276S, NEB) , 12 ⁇ l 10X PAP Buffer, 2.4 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) , 1.2 ⁇ l SUPERase ⁇ In TM RNase Inhibitor (cat. no. AM2694, Thermofisher) , 1.2 ⁇ l Protector RNase Inhibitor (cat. no. 3335399001, Roche) and 12 ⁇ l of 10mM ATP, at 37°C for 15min.
  • A Polymerase
  • 12 ⁇ l 10X PAP Buffer 2.4 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) , 1.2 ⁇ l SUPERase ⁇ In TM RNase Inhibitor (cat. no. AM2694
  • the buffer was then removed, and the cells were rinsed twice with 200 ⁇ l DPBS (cat. no. 14040117, Thermofisher) containing 1%Recombinant RNase Inhibitor (cat. no. 2313, Takara) .
  • adherent cells (either HeLa cells growing on confocal dishes or tissue sectioned on slide) were dehydrated with 100%methanol for 20 min at -20°C, and then rehydrated with three washes of DPBS (cat. no. 14040117, Thermofisher) containing 1%Recombinant RNase Inhibitor (cat. no. 2313, Takara) . After supernatant removal, the cells were blocked by 200 ⁇ l blocking solution containing 175 ⁇ l DPBS (cat. no. 14040117, Thermofisher) , 20 ⁇ l formamide (cat. no.
  • F7503-100ML, Sigma 10 ⁇ l of 20X SSC (cat. no. 15557044, Thermofisher) , 1 ⁇ l of 10 mg/mL Yeast tRNA (cat. no. AM7119, Thermofisher) , 2 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) and 2 ⁇ l of 100uM FAM-ME-Rev oligo.
  • the hybridization was performed at 55°C for 5 min and then 40°C for 2 hours in humid chamber box. After three washes with 0.5 mL washing buffer containing 400 ⁇ l H 2 O, 50 ⁇ l formamide (cat. no. F7503-100ML, Sigma) and 50 ⁇ l of 20X SSC (cat. no. 15557044, Thermofisher) , the cells were ready for microscope imaging.
  • in situ reverse transcription was performed using commercially available reverse transcription reagents and conditions.
  • in situ reverse transcription was performed by adding 120 ⁇ l of reverse transcription mix containing 24 ⁇ l of 10 ⁇ M TruseqRD1_oligo_dT primer (primer can be replaced with TruseqRD2_oligo_dT, TruseqRD1_polyN, TruseqRD2_polyN, TruseqRD2S-polyN if needed) , 21.6 ⁇ l H 2 O, 6 ⁇ l Reverse transcriptase (cat. no.
  • Thermolabile Exonuclease I (cat. no. M0568, NEB) was added to the mixture and incubated at 37 °C for 15 min.
  • the cDNA was further purified using a Zymo DNA Clean and Concentrator-5 Kit (cat. no. D4014, Zymo) by adding 5X volume of DNA binding buffer to the reverse transcription mixture.
  • the DNA was eluted in 21 ⁇ l elution buffer and store at -20°C until ready to amplify.
  • BMKMANU S1000 Spatial Transcriptome Chip BMKGENE
  • All reagents used in spatial barcoding experiment were supplied in BMKMANU S1000 Kit.
  • the tissue section (10 ⁇ m thick) of mouse brain was mounted onto the capture area of BMKMANU S1000 Spatial Transcriptome Chip, and was further subjected to in situ tagmentation, in situ transcription and in situ polyadenylation. After that, the section was dehydrated with 100%methanol for 20 min at -20°C. The slide was further centrifuged at 250 rpm to remove the methanol on the surface.
  • the section was stained at RT for 5 min with 100 ⁇ l staining buffer containing 97.5 ⁇ l 5x SSC (cat. no. 15557044, Thermofisher) , 0.5 ⁇ l Qubit TM ssDNA Reagent (cat. no. Q10212, Thermofisher) , 0.5 ⁇ l Recombinant RNase Inhibitor (cat. no. 2313, Takara) , 0.5 ⁇ l SUPERase ⁇ In TM RNase Inhibitor (cat. no. AM2694, Thermofisher) and 1 ⁇ l Protector RNase Inhibitor (cat. no. 3335399001, Roche) .
  • the section was rinsed once with 100 ⁇ l 0.1X SSC (cat. no. 15557044, Thermofisher) with 1%Recombinant RNase Inhibitor (cat. no. 2313, Takara) .
  • the slide was placed on the dry baths with the active surface facing up and incubated for 1 min at 37°C. Imaging (brightfield and RFP fluorescent channel) was carried out using EVOS TM M7000 (Thermofisher) .
  • permeabilization was performed at 37°C for 6 min with 120 ⁇ l permeabilization buffer containing 20 ⁇ l Protective Agent I, 10 ⁇ l Permeabilization Enzyme and 90 ⁇ l 0.1N HCl. After permeabilization, the section was rinsed by 100 ⁇ l 0.1X SSC (cat. no. 15557044, Thermofisher) with 1%Recombinant RNase Inhibitor (cat. no. 2313, Takara) .
  • reverse transcription was performed at 42°C for 2h with 100 ⁇ l reverse transcription buffer containing 19 ⁇ l H 2 O, 26 ⁇ l RT Reagent, 10 ⁇ l Reducing Agent, 20 ⁇ l In Reagent and 10 ⁇ l Strand-Switching Primer. The supernatant was removed after first-strand synthesis.
  • the chip was incubated at RT with 150 ⁇ l 0.08M KOH (cat. no. P4494-50ML, Thermofisher) for 5min, and then rinsed with 200 ⁇ l Buffer EB (cat. no. 19086, QIAGEN) .
  • second-strand synthesis was performed by adding 100 ⁇ l mixture containing 63 ⁇ l H 2 O, 18 ⁇ l Second-Strand Reagent, 5 ⁇ l Second-Strand Primer, 4 ⁇ l Second-Strand Enzyme and 10 ⁇ l S7-ME oligo (10 ⁇ M) .
  • the second-strand synthesis is carried out at 65 °C for 30 min.
  • the supernatant was removed after second-strand synthesis via reverse transcription.
  • the chip was incubated with 200 ⁇ l Buffer EB (cat. no. 19086, QIAGEN) for 2min, and the supernatant was removed.
  • Buffer EB cat. no. 19086, QIAGEN
  • the second-strand cDNAs (51 ⁇ l in total) were mixed with 0.5 ⁇ l 100 ⁇ M Nextera indexed i7 primer (N701 for example) , 0.5 ⁇ l 100 ⁇ M Truseq indexed i5 primer (NEBNext i501 for example) .
  • a volume of 50 ⁇ l AMP mix (supplied in BMKMANU S1000 Kit) was further added and mixed.
  • the sample was placed in a thermocycler with a heated lid using the following cycling conditions: 95 °C for 30 s; 11 cycles of 95 °C for 30s, 62 °C for 30s and 65 °C for 4 min; final extension at 65 °C for 2 min and hold at 8 °C.
  • Post-PCR clean-up was performed by adding 0.8 ⁇ volume of Ampure XP beads (cat. no. A63881, Beckman Counter) . The PCR mixture was incubated with beads for 15 min at RT, washed twice gently in 80%ethanol, and eluted in 30 ⁇ l Buffer EB (cat. no. 19086, QIAGEN) .
  • the cDNAs (20 ⁇ l in total) were mixed with 2.5 ⁇ l 10 ⁇ M Nextera indexed i7 primer (N702 for example) , 2.5 ⁇ l 10 ⁇ M Truseq indexed i5 primer (NEBNext i502 for example) .
  • a volume of 25 ⁇ l Q5 Hot Start HiFi PCR Master Mix (cat. no. M0543S, NEB) was further added and mixed.
  • the sample was placed in a thermocycler with a heated lid using the following cycling conditions: 95 °C for 30 s; 11 cycles of 95 °C for 30s, 62 °C for 30s and 65 °C for 1 min; final extension at 65 °C for 2 min and hold at 8 °C.
  • Post-PCR clean-up was performed by adding 0.8 ⁇ volume of Ampure XP beads (cat. no. A63881, Beckman Counter) .
  • the PCR mixture was incubated with beads for 15 min at room temperature (RT) , washed twice gently in 80%ethanol, and eluted in 30 ⁇ l Buffer EB (cat. no. 19086, QIAGEN) .
  • the eluted DNA (20 ⁇ l in total) was mixed with 2.5 ⁇ l 10 ⁇ M Nextera indexed i7 primer (N703 for example) , 2.5 ⁇ l 10 ⁇ M Nextera indexed i5 primer (N503 for example) .
  • a volume of 25 ⁇ l Q5 Hot Start HiFi PCR Master Mix (cat. no. M0543S, NEB) was added and mixed.
  • the sample was placed in a thermocycler with a heated lid using the following cycling conditions: 65 °C for 5 min; 95 °C for 30 s; 11 cycles of 95 °C for 30s, 62 °C for 30s and 65 °C for 1 min; final extension at 65 °C for 2 min and hold at 8 °C.
  • Post-PCR clean-up was performed by adding 0.95 ⁇ volume of Ampure XP beads (cat. no. A63881, Beckman Counter) . The PCR mixture was incubated with beads for 15 min at RT, washed twice gently in 80%ethanol, and eluted in 30 ⁇ l Buffer EB (cat. no. 19086, QIAGEN) .
  • SeekSpace is a commercial spatial platform resembling Slide-tags (see Russell, A.J.C., et al. (2024) , Slide-tags enables single-nucleus barcoding for multimodal spatial genomics, Nature 625, 101–109) , which is used to perform spatial barcoding of the in situ transcripts.
  • a tissue section is applied to a SeekSpace slide of barcoded oligos that have been spatially indexed.
  • the tissue section on the SeekSpace slide is subjected to in situ tagmentation and in situ transcription.
  • the DNA spatial barcodes associate with nuclei, allowing spatially barcoded nuclei to be profiled via established droplet-based single-nucleus sequencing, such as SeekOne.
  • droplet-based single-nucleus sequencing such as SeekOne.
  • T 1: 1 ratio mixed poly
  • N poly
  • the Visium CytAssist is a commercial instrument designed to facilitate the transfer of oligonucleotides (such as transcriptomic probes) from standard glass slides to Visium spatial slides.
  • in situ tagmentation is performed with T7-HandleA-ME /ME-Rev Tn5, followed by in situ transcription.
  • in situ reverse transcription is performed with TruseqRD2S-polyN.
  • an RNA digestion step is performed using 1X RNase H Reaction Buffer containing RNase H (NEB, M0297S) at 37°C for 20 min.
  • oligonucleotides HandleB-polyA, Splint-2G, Splint-3G, Splint-4G were dissolved in water to a final concentration of 100 ⁇ M each. These oligos were mixed in a 5: 2: 2: 1 ratio and denatured for 5 min at 95 °C and cooled down slowly (-0.1 °C/sto 16 °C) for annealing using a thermocycler.
  • DBiT-seq combines microfluidic deterministic barcoding with next-generation sequencing (NGS) to simultaneously profile RNA and protein expression while preserving spatial context.
  • NGS next-generation sequencing
  • in situ tagmentation is performed with T7-S5-ME /ME-Rev Tn5 or Sp6-S5-ME/ME-Rev Tn5, followed by in situ transcription.
  • PDMS chips were ordered from FluidicLab.
  • DBiT-seq was performed after in situ trancription according to the protocol described in High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183.6 (2020) : 1665-1681 but omitting the step of template switch. To visualize the channel, FITC-conjugated BSA was spiked in.
  • Dissociated fresh HeLa cells were subjected to standard ATAC-seq. Directly following transposition, nuclei were spun down (800 g at 4 °C for 5 min) , and washed twice with 200 ⁇ l of ice-cold DPBS. Then nuclei were resuspended in 200 ⁇ l PBS containing 1 ⁇ g/ ⁇ l and transferred to a FACS tube.
  • DAPI positive single nuclei were sorted into each well in a 96-well plate containing 3 ⁇ l of lysis buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.2%SDS, 10 ⁇ M Nextera indexed i7 primer and 10 ⁇ M Nextera indexed i5 primer) and incubated at 65 °C for 15 min.
  • lysis buffer 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.2%SDS
  • 10 ⁇ M Nextera indexed i7 primer and 10 ⁇ M Nextera indexed i5 primer were incubated at 65 °C for 15 min.
  • Nextera i7 primers were indexed with single-cell barcodes
  • Nextera i5 primers were indexed with plate barcodes.
  • 1 ⁇ l of 10%tween-20 was added per well to quench SDS, followed by adding 4 ⁇ l of Q5 High-Fidelity 2 ⁇ Master Mix (cat. no.
  • in situ tagmentation in situ T7 transcription, in situ polyadenylation and in situ reverse transcription was performed inside nuclei of dissociated HeLa cells after fixation. Centrifugation steps (800 g at 4 °C for 5 minutes) were conducted to remove supernatant when necessary. After in situ reverse transcription, nuclei were stained by DAPI and sorted to 96-well plate containing 3 ⁇ l of lysis buffer as descripted above. Notably, Nextera i5 primers were replaced by Truseq i5 primers indexed with plate barcodes. Nextera i7 primers with single-cell indexes were used to barcode cDNA of transcripts in each cell. The amplification and post-PCR purification was the same with standard single cell experiments.
  • Fig. 1 is a schematic depicting a method of spatial profiling of open chromatin.
  • Cells on dishes or tissue sections on slides were fixed and permeabilized. Permeabilized nuclei were incubated in situ with transposome containing RNA polymerase promoter sequence and transposase binding sequence (for Tn5, the binding sequence is Mosaic End, ME) .
  • the accessible regions of genome were fragmented and tagged with T7 promoter sequence via transposon.
  • RNA polymerase was subsequently used for in situ transcription to amplify the genomic DNA fragments into genomic RNAs.
  • the design of the transposome ensures the presence of ME sequence in the beginning region of RNA transcripts, enabling their labeling with fluorescent probes.
  • enzymatic in situ polyadenylation was performed on in situ transcripts, which facilitates the transcripts being compatible with reverse transcription and spatial barcoding.
  • Fig. 2 is a representative image of the transcripts inside nucleus after in situ tagmentation and in situ transcription.
  • Panel A HeLa cells on dishes were subjected to in situ tagmentation (utilizing T7-ME/ME-Rev Tn5) and in situ transcription inside nucleus (stained with DAPI) . Fluorescent FAM-ME-Rev probes were used for the hybridization with transcripts. Compared with the negative control (in situ tagmentation only) , in situ transcription leads to the accumulation of transcripts within nucleus.
  • Fig. 3 is a schematic illustration of spatial profiling of histone modifications.
  • Cells on dishes or tissue section on slides were fixed and permeabilized.
  • Transposase Tn5 was fused with an antibody binding domain Protein A/G.
  • Permeabilized nuclei were first incubated in situ with antibodies targeting histone modifications, followed by incubation with pAG-Tn5 transposome (T7-ME/ME-Rev pAG-Tn5) .
  • the antibodies bound to histone modifications mediated the recruitment of pAG-Tn5 transposome, leading to fragmentation and tagmentation of genomic DNA nearby targeted histone modifications.
  • in situ transcription and in situ polyadenylation enable transcripts to adopt a mRNA-like structure, ensuring compatibility with reverse transcription and spatial barcoding.
  • Fig. 4 is representative imaging of transcripts inside nucleus after in situ antibody directed tagmentation and in situ transcription.
  • Upper row HeLa cells on dishes were subjected to in situ antibody directed tagmentation (using T7-ME/ME-Rev Tn5) and in situ transcription inside nucleus (stained with DAPI) .
  • a very limited in situ transcript signal is observed in Normal Rabbit IgG negative control.
  • Lower row In contrast, significant fluorescence within the nuclei was observed in the anti-H3K4me3 antibody group and the anti-H3K27ac antibody group, indicating that antibodies binding to targeted histone modifications promote proximal tagmentation.
  • Fig. 5 is representative imaging of the transcripts inside nucleus after in situ tagmentation and in situ transcription with different RNA polymerases.
  • In situ tagmentation was conducted using Tn5 transposomes containing T3-S7-ME/ME-Rev, Sp6-S7-ME/ME-Rev, and T7-S7-ME/ME-Rev. Subsequently, corresponding T3 RNA polymerase, Sp6 RNA polymerase, and T7 RNA polymerase were utilized in the in situ transcription step. Each enzyme yielded a higher fluorescent signal compared to the negative control group, with T7 exhibiting the strongest signal, followed by Sp6, and T3. DAPI staining was used to determine the position of nuclei.
  • Fig. 6 is representative imaging of the in situ transcripts inside nucleus after in situ tagmentation with Tn5 containing different transposon sequences.
  • the flanking sequences of the T7 promoter were altered to assess their impact on transcription efficiency. Replacing the sequence in front of the T7 promoter (T7-S7-ME vs T7-S7-ME2) did not yield any noticeable effects. Using sequences with three G bases following the T7 promoter, including T7-S7-ME3, T7-S7-ME-4 and T7-3G-S7-ME, resulted in a stronger signal.
  • Fig. 7 depicts genome browser tracks of open-chromatin libraries from Hela cells. Chromatin accessibility data were generated with a library by standard ATAC-seq and produced by in situ tagmentation, in situ transcription, and in situ reverse transcription. Genome locations are indicated at the top of the tracks, and gene names are shown at the bottom. The data profiling of open chromatin with different methods demonstrates a high level of concordance.
  • Fig. 8 depicts data from experiments showing consistent performance across different open-chromatin profiling methods.
  • Panel A Comparison of chromatin accessibility peak overlaps across replicates of each method was conducted by calculating pairwise correlations. The correlation matrix indicates a high level of concordance in the accessible genome mapped by different methods.
  • Panel B Sequencing data obtained through in situ tagmentation, in situ transcription, and in situ reverse transcription exhibited significant enrichment for fragments within transcription start site (TSS) regions, along with a fragment-size distribution similar to that observed in standard ATAC-seq.
  • Panel C Tn5 insertion positions were centered over CTCF motifs in called peaks. The aggregation of insertion sites generated a CTCF binding footprint, indicating a high quality of the dataset.
  • Fig. 9 depicts data from open-chromatin profiling by different methods in single cells.
  • Panel A Comparison of read counts and unique fragment counts in single-cell libraries generated using standard ATAC-seq versus those produced through in situ tagmentation, in situ transcription, and in situ reverse transcription revealed a significant increase relative to standard single-cell ATAC.
  • Panel B A depiction of chromatin accessibility spanning a segment of the human genome in HeLa cells. Tracks include data from bulk standard ATAC-seq library, bulk library generated via in situ tagmentation, in situ transcription, and in situ reverse transcription, as well as an aggregation of single-cell libraries and single-cell libraries created through in situ tagmentation, in situ transcription, and in situ reverse transcription. Open chromatin profiles at both bulk and single-cell levels demonstrates a high degree of concordance.
  • Fig. 10 depicts data from experiments of spatial barcoding of in situ transcripts with spatial transcriptomic slide BMKMANU.
  • Fig. 11 depicts spatial distribution of chromatin accessibility in the hippocampal region of mouse brain on a BMKMANU S1000 Spatial Transcriptome Chip.
  • the density of captured fragments (number of fragments, n_fragment) from each spot (50 ⁇ m) was projected on their spatial location.
  • the hippocampal dentate gyrus, housing neurons with high levels of chromatin accessibility, exhibits a greater number of captured fragments and forms a distinctive landmark structure.
  • Spatial mapping of the gene activity score of the hippocampal pyramidal cell marker Cnksr2 and hippocampal neuron marker Epha7 reveals patterned accessibility, including enrichment in hippocampal region.
  • the gene activity score non-specific gene Gapdh in the same region was mapped as control.
  • Fig. 12 is a schematic illustration of spatial profiling of in situ transcripts using Slide-tags strategy.
  • the oligos with spatial barcodes are cleaved and diffuse into tissue section to associate them with nuclei.
  • Nuclei from intact fresh-frozen tissue sections are labeled with positional barcode oligonucleotides derived from spatially indexed slide, followed by the labeling of both in situ transcripts and with single-cell barcode oligonucleotides using standard single-cell techniques.
  • Fig. 13 depicts data from experiments of spatial barcoding of in situ transcripts with spatial transcriptomic platform SeekSpace.
  • Fig. 14 is a schematic illustration of spatial profiling of in situ transcripts using Visium CytAssist.
  • the in situ tagmentation, in situ transcription, in situ reverse transcription with poly (N) probe, and in situ ligation of cDNA with a polyA tailed adaptor was performed on the tissue section attached to standard glass slide.
  • the standard glass slide with tissue section and a Visium slide with capture area are loaded into the CytAssist instrument.
  • the system precisely aligns the tissue section from the standard slide onto the corresponding capture areas for capturing diffusing cDNA.
  • the cDNA of in situ transcripts are indexed with spatial barcodes on Visium slide.
  • Fig. 15 depicts data from experiments of spatial barcoding of in situ transcripts with Visium CytAssist.
  • Panel A) A brightfield image is captured to provide spatial localization and orientation of mouse brain section corresponding to the Visium slide within the CytAssist instrument.
  • Panel B) The density of captured fragments (number of fragments, n_fragment) from each spot (single-cell) was projected on their spatial location.
  • Fig. 16 depicts data from open-chromatin profiling of mouse brain by different methods. Tracks include data in the region of two representative genomic regions for visual comparison. On Chr1 (175, 554, 488-175, 877, 638) and Chr1 (88, 120, 158-88, 443, 308) , detection of in situ transcripts with BMKMANU, SeekSapce and Visium produce identical open chromatin sharp peaks with a strong signal-to-noise ratio, a pattern also shown by standard DBiT-seq ATAC data from publication (Deng, Y., Bartosovic, M., Ma, S. et al. (2022) Spatial profiling of chromatin accessibility in mouse and human tissues. Nature 609, 375–383. ) .
  • Fig. 17 is a schematic illustration of spatial profiling of in situ transcripts using DBiT-seq strategy.
  • a PDMS microfluidic chip 1 (50 parallel channels, 20–50 ⁇ m wide) is clamped onto the slide, enabling sequential barcoding.
  • DNA barcode A poly (T) probes or poly (N) probes with spatial barcode Ai (A1 to A50)
  • Reverse transcription incorporates barcode A into cDNA of in situ transcripts.
  • a second PDMS chip 2 (channels perpendicular to the first) introduces barcode B (spatial barcode Bi (B1 to B50) and PCR handle) .
  • barcode B spatial barcode Bi (B1 to B50) and PCR handle
  • T4 ligation links barcodes A and B at channel intersections, spatially creating a 50 ⁇ 50 barcode mosaic.
  • Fig. 18 depicts data from experiments of spatial barcoding of in situ transcripts with DBiT-seq.
  • Panel A Fluorescence imaging of in situ transcripts (labeled with FITC-UTP during transcription) , marking regions of transcriptionally active chromatin across the embryo.
  • Panel B The density of cDNA unique fragments (number of fragments, n_fragment) from each spot (50 ⁇ m) was projected on their spatial location.
  • Panel C Comparison of unique fragment counts in spatial chromatin accessibility detection of mouse embryo using standard DBiT-seq ATAC data from publication (Deng, Y., Bartosovic, M., Ma, S. et al. (2022) Spatial profiling of chromatin accessibility in mouse and human tissues. Nature 609, 375–383. ) versus those produced through in situ tagmentation and in situ transcription revealed a significant increase relative to standard spatial ATAC with DBiT-seq platform.
  • Fig. 19 depicts data from experiments showing spatial maps of gene activity scores of mouse embryo, demonstrating region-specific chromatin accessibility.
  • Hoxb3 a homeobox gene critical for anterior-posterior patterning, exhibited spatially restricted activation along the body axis.
  • Runx2 a master regulator of early tooth development, showed high accessibility in regions corresponding to in the dental mesenchyme.
  • FIG. 20 depicts data from experiments showing spatial maps of transcription factor activities of mouse embryo, illuminated region-specific regulation. Gata4, essential for cardiac development, displayed enriched activity within regions consistent with forming cardiac tissues.
  • Pou4f2 associated with neurogenesis, demonstrated strong activity in neurogenic domains of the embryonic spinal cord.
  • Fig. 21 depicts data from experiments uncovering region-specific chromatin accessibility patterns by clustering analysis.
  • Panel A UMAP unsupervised projection of individual spots based on chromatin accessibility-derived gene activity, revealing distinct clusters.
  • Panel B Spatial mapping of UMAP clusters onto the embryonic tissue section, illustrating the anatomical organization of regulatory domains.
  • Fig. 22 depicts data from experiments showing spatially resolved open chromatin profiles of across distinct tissue clusters. Normalized signal intensity curves (range: 0–99) for six spatial clusters (0–6, color-coded in accordance with UMAP clusters) along the genomic region on chromosome 11 (chr11: 96,320,000–96,350,000 bp) covering Hoxb3 gene. Each curve represents the aggregate fragments showing cluster-specific signal fluctuations (relatively increased in cluster 0, 1 and 3) .
  • kits of the present disclosure generally will include one or more of at least a nuclease or transposase, transcriptase, polyadenylase (Poly (A) polymerase) , degradation enzyme, nucleotides, DNA ligase, DNA polymerase, amplification primers and reagents, sequencing primers and reagents, and/or DNA enrichment reagents described herein which may be used to carry out the claimed method.
  • the kit will also contain directions for treating chromatin DNA with a nuclease or transposase system, a transcriptase and polyadenylase (Poly (A) polymerase) processing and amplifying the treated chromatin DNA.
  • the kits will preferably have distinct containers for each individual reagent, enzyme or reactant. Each agent will generally be suitably aliquoted in their respective containers.
  • the container means of the kits will generally include at least one vial or test tube. Flasks, bottles, and other container means into which the reagents are placed and aliquoted are also possible.
  • the individual containers of the kit will preferably be maintained in close confinement for commercial sale. Suitable larger containers may include injection or blow-molded plastic containers into which the desired vials are retained. Instructions are preferably provided with the kit.
  • the present disclosure provides a method of analyzing genomic DNA of a cell including the steps of (a) in situ fragmenting genomic DNA within a cell to create a plurality of genomic DNA fragments, (b) in situ transcribing the genomic DNA fragments into corresponding RNA fragments to create a plurality of RNA fragments, (c) in situ polyadenylating the plurality of RNA fragments to create a plurality of polyadenylated RNA fragments having a poly (A) tail, (d) capturing the plurality of polyadenylated RNA fragments using a plurality of capture oligonucleotide probes to create a plurality of captured polyadenylated RNA fragments, and (e) reverse transcribing the plurality of captured polyadenylated RNA fragments to create a plurality of cDNA fragments having barcodes.
  • the present disclosure provides a method of analyzing genomic DNA of a cell including the steps of (a) in situ fragmenting genomic DNA within a cell to create a plurality of genomic DNA fragments, (b) in situ transcribing the genomic DNA fragments into corresponding RNA fragments to create a plurality of RNA fragments, (c) in situ polyadenylating the plurality of RNA fragments to create a plurality of polyadenylated RNA fragments having a poly (A) tail, (d) capturing the plurality of polyadenylated RNA fragments using a plurality of capture oligonucleotide probes to create a plurality of captured polyadenylated RNA fragments, (e) reverse transcribing the plurality of captured polyadenylated RNA fragments to create a plurality of cDNA fragments, and (f) performing second strand synthesis to create products containing genomic RNA information and with barcodes.
  • the method further includes sequencing the plurality of cDNA fragments. According to one aspect, the method further includes amplifying the plurality of cDNA fragments. According to one aspect, the method further includes amplifying and sequencing the plurality of cDNA fragments. According to one aspect, steps (a) , (b) , and (c) are performed on a plurality of cells. According to one aspect, steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample. According to one aspect, steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample and the tissue sample is contacting a substrate comprising oligonucleotide capture probes.
  • step (d) is carried out by lysing the plurality of cells and the plurality of polyadenylated RNA fragments bind to the oligonucleotide capture probes on the substrate.
  • step (a) is carried out by a transposition system to create tagged DNA fragments.
  • step (a) is carried out by nuclease digestion to create DNA fragments.
  • the transposition system is a Tn5 transposition system.
  • the transposition system is a MuA transposition system.
  • step (b) is carried out using a T7 RNA polymerase, T3 RNA polymerase, or Sp6 RNA polymerase.
  • the cell is a single cell and the plurality of isolated polyadenylated RNA fragments of step (d) corresponds to the single cell.
  • steps (a) , (b) , and (c) are performed on a plurality of cells of a tissue sample and the plurality of isolated polyadenylated RNA fragments of step (d) comprises polyadenylated RNA fragments corresponding to each of the plurality of cells.
  • the oligonucleotide capture probes include oligo (dT) sequences.
  • the oligonucleotide capture probes include sequences acting as random primer.
  • the oligonucleotide capture probes include sequences acting as sequence specific probes.
  • steps (e) and (f) use barcodes capable of indexing spatial positions.
  • steps (e) and (f) use barcodes capable of indexing single cells.
  • steps (e) and (f) use barcodes capable of indexing single cells and spatial positions.
  • the genomic DNA fragments include one or more open regions.
  • the genomic DNA is processed to enrich for open chromatin DNA.
  • the genomic DNA fragments include one or more targeted regions.
  • the genomic DNA fragments include one or more targeted regions enriched for histone modifications.
  • the genomic DNA fragments include one or more targeted regions enriched for histone modifications and transcription factor binding sites. According to one aspect, the genomic DNA fragments include one or more targeted regions enriched for histone modifications, transcription factor binding sites and DNA modifications. According to one aspect, the genomic DNA fragments include one or more targeted regions enriched for cytosine converted to uracil by deamination. According to one aspect, the genomic DNA fragments comprise one or more targeted regions enriched for DNA methylation.

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Abstract

L'invention concerne des procédés d'analyse d'ADN génomique où l'ADN génomique est soumis à une fragmentation in situ, une transcription in situ, une synthèse de l'ADN complémentaire et un codage à barres de l'ADN complémentaire.
PCT/CN2025/095434 2024-05-17 2025-05-16 Procédés d'analyse épigénomique spatiale dans des cellules individuelles d'échantillon de tissu Pending WO2025237407A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
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WO2019213294A1 (fr) * 2018-05-03 2019-11-07 Becton, Dickinson And Company Analyse multi-omique d'échantillons à haut débit
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