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WO2019116306A1 - Système de capture ou d'immunoprécipitation d'un complexe protéine-adn - Google Patents

Système de capture ou d'immunoprécipitation d'un complexe protéine-adn Download PDF

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WO2019116306A1
WO2019116306A1 PCT/IB2018/060023 IB2018060023W WO2019116306A1 WO 2019116306 A1 WO2019116306 A1 WO 2019116306A1 IB 2018060023 W IB2018060023 W IB 2018060023W WO 2019116306 A1 WO2019116306 A1 WO 2019116306A1
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previous
protein
microfluidic
chip
dna
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Bart Deplancke
Riccardo DAINESE
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Ecole Polytechnique Federale de Lausanne EPFL
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Ecole Polytechnique Federale de Lausanne EPFL
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention concerns a microfluidic device for rapid, multiplexed, bead-less chromatin immunoprecipitation, herein named FloChIP.
  • the microfluidic device may additionally include on-chip DNA library preparation.
  • the present invention concerns a system or method for capture or immunoprecipitation of a protein-DNA complex, for example, chromatin immunoprecipitation of histone marks or transcription factors.
  • ChIP-seq Chromatin immunoprecipitation followed by sequencing
  • ChIP-seq has been fruitfully performed on different organisms, including mouse and humans [2-4], and has been adopted by renowned international consortia like the ENCODE project [4] As of October 2017, ChIP-seq has been cited in more than 17 ⁇ 00 PubMed scientific articles.
  • ChIP-seq Since its introduction in 2007, ChIP-seq has been modified in different ways [5-7]; however, these modifications have mainly addressed the pre-immunoprecipitation preparation of chromatin. On the other hand, solid-state functionalized beads, specific antibodies and DNA library preparation have remained a constant across all the different ChIP-seq implementations.
  • ChIP-seq is an intensive manual protocol, requiring multiple steps, reagents, consumables and expensive antibodies [8] This weighs on laboratories budgets and personnel time, thus reducing the possible scope and scale of experiments.
  • SX-8G IP-Star ® Compact Automated System by Diagenode 9
  • the IP-star is a bulky semi-automated robot which still requires a substantial amount of consumables and reagents.
  • ChIP-seq Another important bottleneck for current ChIP-seq implementations is the quantity of cellular material needed in order to produce high quality sequencing data. Both manual and the above-mentioned automated ChIP-seq require millions of cells, thus limiting the application of the protocol to scarce but medically relevant samples like small tumors and tissue biopsies [10].
  • ChIP-seq Chromatin immunoprecipitation followed by next generation sequencing
  • the ENCODE and modENCODE consortia have performed more than 8,000 ChIP-seq experiments, which have enhanced our collective understanding of how gene regulatory processes are orchestrated in humans as well as several model organisms.
  • ChIP-seq proved to be essential to acquire new insights into genomic organization and into the mechanisms underlying genomic variation-driven phenotypic diversity and disease susceptibility. More specifically, this assay proved crucial in determining the DNA binding properties of hundreds of TFs. Nevertheless, in comparison to other widespread NGS-based methods - e.g. RNA-seq, ATAC-seq, and Hi- C - ChIP-seq lags behind in some key metrics, i.e. throughput, sensitivity, modularity, and automation, which hinder its wider adoption and reproducibility.
  • RNA-seq can now be regularly performed on hundreds or thousands of single cells using readily available workflows
  • ChIP-seq has largely remained labor intensive and limited to few samples per run, each composed of millions of cells.
  • a typical pre-amplification RNA-seq workflow consists of only three steps - i.e. cell lysis, RNA capturing and reverse transcription - ChIP-seq typically involves several pre-amplification steps (crosslinking, lysis, fragmentation, immunoprecipitation, end-repair and adapter ligation).
  • any given RNA transcript is present in each cell in numerous copies, which increases the likelihood of its capture and detection, whereas, on the other hand, each locus-specific protein-DNA contact occurs a maximum of two times in a diploid cell.
  • the combination of these idiosyncratic differences, together with the lack of enabling solutions, has thus far prevented the ChIP-seq technology, as opposed to other NGS-based methods, to reach its full potential in terms of adoption and overall utility.
  • sequential-ChIP In addition to the standard ChIP protocol, a modification of its workflow involving sequential chromatin immunoprecipitation (sequential-ChIP) has also been adopted to infer genomic co-occurrence of two distinct protein targets.
  • sequential-ChIP consists in performing ChIP twice on the same input chromatin, which leads to a multiplication of the inefficiencies mentioned above. Therefore, not only does sequential-ChIP show the same limitations as regular ChIP-seq, but these also come in an augmented form due to its sequential nature. As a result, despite the multi-dimensional information provided, sequential- ChIP has also resisted wider adoption.
  • Ma et al.[18] and Rotem et al. [19] addressed the limits of sensitivity with two different microfluidic-based strategies.
  • Ma et al. focused on improving the efficiency of the IP step by confining it within microfluidic channels. Although these researchers showed good IP efficiency down to as few as 30 cells, their approach requires impractical antibody-oligo conjugates, is not automated and was not shown to work for TFs.
  • Rotem et al. achieved the remarkable feat of performing ChIP-seq in a single cell by integrating the concept of chromatin barcoding and pooling into a single droplet-based microfluidic chip.
  • the barcoding step has indeed single cell resolution
  • the most critical step - i.e. the IP step - is performed manually on 100 cells.
  • their approach which was also shown to work only for histone marks so far, yielded sparse single cell data and thousands of assays are needed to identify specific cell subpopulation signatures.
  • the present invention addresses the above-mentioned limitations by providing a system for immunoprecipitation of a protein-DNA complex or for capture of a protein-DNA complex according to claim 1 and a method for immunoprecipitation of a protein-DNA complex or for capture of a protein-DNA complex according to claim 40.
  • FloChIP microfluidic system
  • the device, system and method of the present invention shows that high quality one-day and parallelized ChIP-seq for histone marks (down to 500 cells and thus highly-sensitive) and transcription factors TFs (100 ⁇ 00 cells) can be achieved through a combination of microvalves, microstructures, flexible surface chemistry and on-chip chromatin tagmentation.
  • the interconnected and modular device configuration of FloChIP enables straightforward re- immunoprecipitation of eluted chromatin, effectively enabling sequential-ChIP which allows to probe bivalent chromatin with unprecedented ease.
  • the FloChIP has a wide dynamic input range (immunoprecipitation from 10 s to 500 cells) and it faithfully reproduces ENCODE data for all tested histone marks (immunoprecipitation on H3k27ac, H3k4me3, H3k4mel, H3k27me3 and H3k9me3).
  • the high-throughput capabilities of the present invention are shown by performing ChIP-seq for a transcription factor (MEF-2A) on chromatin derived from 32 distinct lymphoblastoid cell lines.
  • MEF-2A transcription factor
  • Figures 1A and IB show exemplary systems and devices of the present disclosure.
  • Figure 2 schematically shows an exemplary microfluidic device or chip of the present disclosure.
  • Figure 3 schematically shows exemplary elements of the microfluidic device or chip of the present disclosure.
  • Figure 4 is a schematic a cross-sectional view of an exemplary surface biofunctionalization of a surface in a channel of an antibody unit of the microfluidic device or chip of the present disclosure.
  • Figures 5a to 5f show data obtained using the system and method of the present disclosure performed on different chromatin-associated protein modifications namely, H3k27ac, H3k4me3, H3k4mel, H3k27me3 and H3k9me3 and this data is compared with publicly available ChIP-seq data generated by the ENCODE project.
  • Figures 6a and 6b presents data related to lower detection limit measurements of the system of the present disclosure.
  • Figure 6c presents a table containing data related to known ChIP-seq systems.
  • Figure 7a to 7e generally show the microfluidic device architecture for miniaturized ChIP-seq.
  • Figure 7a shows exemplary processing phases in descending chronological order of the system and method of the present disclosure.
  • the inner walls are functionalised by sequentially introducing chemical species that firmly interact with both the previous and following layer of functionalization.
  • these species can be, for example, biotin-BSA, neutravidin, biotin-Protein A/G and antibody.
  • IP takes place by flowing sonicated chromatin on-chip in a total time of 30-60 minutes, depending on the chromatin volume introduced.
  • the antibody-bound chromatin is tagmented directly on-chip in order to introduce lllumina-compatible adapters.
  • the tagmented chromatin is eluted off-chip by use of a SDS-containing buffer and high temperature.
  • Figure 7b shows a top-view of a portion containing numerous micro-pillars. Each portion is itself repeated several times along the length of one IP lane.
  • FIG. 7c shows a top-view schematic of one IP lane.
  • Each IP lane can be repeated n times across a FloChIP device and includes flow channels and control channels.
  • Figure 7d shows fluorescence micrographs showing biotin-BSA in the correct formation of the FloChIP's totem.
  • Figure 7e shows a top-view schematic of a high-throughput 64-unit FloChIP device, including flow channels and control channels.
  • Figures 8a to 8h generally concern the system's robust generation of chromatin landscapes for histone marks.
  • Figure 8a shows a schematic depiction of FloChIP's mode 1: antibody multiplex. Each IP lane is functionalized separately by introducing different antibodies through the individual inlets. During IP, one sample is introduced through the common inlet and distributed equally across all IP lanes.
  • Figure 8b is a schematic depiction of FloChIP's mode 2: sample multiplex.
  • One antibody solution is introduced through the common inlet and distributed equally across all IP lanes. During IP, each IP lane is loaded separately by introducing different samples through the individual inlets.
  • Figure 8c shows signal tracks for FI3k27ac, FI3k4mel and FI3k4me3 profiles obtained by the system of the present disclosure.
  • ENCODE data generated by conventional ChIP-seq are also shown.
  • Figure 8d shows FI3k27ac profiles obtained by the system of the present disclosure with decreasing cell numbers.
  • ENCODE data generated by conventional ChIP-seq are also shown.
  • Figure 8e shows genome-wide correlation plots between FloChIP (x axis) and ENCODE (y axis) data for all targets tested, i.e. FI3k27ac, FI3k4mel, FI3k9me3, FI3k27me3 and FI3k4me3.
  • Figure 8f shows normalized read density profiles around transcription start sites for samples of decreasing cell numbers and ENCODE.
  • Figure 8g shows genome-wide correlation between pairs of samples with decreasing cell numbers
  • Figure 8h shows a comparison in terms of fraction of reads in peaks (FRiP) between the system of the present disclosure and ENCODE for histone mark samples.
  • Figure 9a to 9d general relates to a "sequential IP" mode of the system of the present disclosure for the study of bivalent chromatin.
  • Figure 9a shows exemplary sequential IP steps in descending chronological order for the case of FI3k4me3-FBk27me3. Chromatin coming from the first IP is collected into off-chip reservoirs connected to device. Following collection, the control channels are actuated in a way to isolate the first IP lane from the chromatin, while opening the path to the second IP lane. At this point, the chromatin flown into the second pre-functionalised IP lane. Finally, the bivalent chromatin is eluted again in off-chip reservoirs.
  • Figure 9b shows locus-specific signal tracks for the two individual IP libraries (H3k4me3 and H3k27me3) as well as the corresponding sequential IP samples (H3k27me3/H3k4me3 and FI3k4me3/ H3k27me3).
  • Figure 9c shows a bivalency score distribution for HCP promoters. The shade-codes reflect the relative abundance of the two individual marks for each promoter.
  • Figure 9d shows Gene Ontology enrichment analysis for the first one thousand promoters with the highest bivalency score.
  • Figure 10a to g generally concern transcription factor IP in high-throughput mode performed by the system of the present disclosure.
  • Figure 10a is a list of the 32 cell lines used in this study.
  • Figure 10b shows qPCR enrichment for each library. The average across all libraries log 2 (fold change ) is 5.7.
  • Figure 10c shows the percent of mapped reads for each library. The average mapping rate across all libraries is 46.6%.
  • Figure lOd shows signal tracks reported for each library for three different genomic regions.
  • Figure lOe shows the number of peaks called for each library (3374 peaks on average).
  • Figure lOf shows the FRiP score for each library (6.9% on average).
  • Figure lOg shows the MEF-2A motif enrichment for each library (a -log(Pvalue) of 9.2 on average).
  • Figure 11a also shows exemplary processing phases of the system and method of the present disclosure in descending chronological order in the case of chromatin/antibody pre-incubation.
  • the inner walls are functionalised by sequentially introducing chemical species that firmly interact with both the previous and following layer of functionalization. Also in chronological order, these species are for example biotin-BSA, neutravidin, biotin-Protein A/G.
  • the pre-incubated antibody/chromatin is flown on-chip in a total time of 30- 60 minutes, depending on the volume introduced.
  • the antibody-bound chromatin is tagmented directly on-chip in order to introduce lllumina-compatible adapters.
  • the tagmented chromatin is eluted off-chip by use of a SDS-containing buffer and high temperature.
  • Figure lib is a schematic of exemplary elements of the system of the present disclosure.
  • Figure 11c is an example of a COMSOL simulation used to optimise device architecture.
  • Figure lid is a top-view schematic of the medium-throughput 8-unit microfiuidic device of the present disclosure including flow channels and control channels.
  • Figure lie shows an exemplary microfiuidic 64-outlet multiplexer for pressure distribution into microfiuidic device of the present disclosure
  • Figure Ilf shows an exemplary 16-outlet multiplexer.
  • Figures 12a to 12f concern results for IP on histone marks.
  • Figure 12a shows amplification cycle statistics for samples of decreasing ceil number, from 1 million to 500 ceils.
  • Figure 12b shows fold enrichment statistics for samples of decreasing cell number, from 1 million to 500 ceils.
  • Figure 12c shows a mapping rate for samples of decreasing ceil number, from IQO'OOQ to 500 ceils.
  • Figure 12d shows a FRiP score for samples of decreasing cell number, from 1 million to 500 cells.
  • Figure 12e shows normalized read density profiles around transcription start sites for H3k4me3, H3k27ac and H3k4mel.
  • Figure 12f shows fold enrichment statistics for histone mark samples, namely H3k4me3, H3k27ac, H3k27me3, H3k9me3 and H3k4mel.
  • Figures 13a and 13b show signal tracks for individual and sequential IP libraries previously reported for the same loci.
  • Figure 13c shows correlation results between results provided by the system and method of the present disclosure and previously published Co-Chip data.
  • Figures 14a and 14b concern genome wide characterization of TF data by the system and method of the present disclosure.
  • Figure 14a shows normalized read density profiles around transcription start sites for all sequenced libraries (thicker line being the average profile).
  • Figure 14b shows correlation results between ail pairs of sequenced libraries.
  • FIGS 1A and IB schematically show an exemplary system 1 of the present disclosure.
  • the system 1 is, for example, a system for capture or immunoprecipitation of a protein-DNA complex; or a protein-DNA complex capture or immunoprecipitation system.
  • the system 1 is capable of supporting microfiuidic, bead-less, automated, scalable, multiplexed and sensitive immunoprecipitation (see, for example, Fig. 1A and IB) This system 1 is called FloChIP herein.
  • the system 1 is a bead-less system or a magnetic bead-less system.
  • the system 1 is, for example, an immunoprecipitation system configured to determine protein/DNA interactions in vivo.
  • the system is, for example, configured to immunoprecipitate or capture a protein- DNA complex.
  • the system 1 is, for example, a chromatin immunoprecipitation system.
  • the system 1 includes a microfluidic device or chip 108 comprising at least one or a plurality of antibody units 111, 201.
  • the antibody unit 111 (or each antibody unit 111) includes at least one microfluidic channel MFC or a plurality of microfluidic channels MFC.
  • the microfluidic channel MFC includes at least one or a plurality of surfaces configured to receive a multi layered assembly MLA of biochemical species (Figure 4).
  • the multi-layered assembly MLA of biochemical species defines or forms a biofunctionalized surface.
  • the biofunctionalized surface is configured to capture a target molecule.
  • the antibody unit 111 contains one or more side walls wl, w2 as well as an upper wall or ceiling CL and a lower wall or floor FL defining the microfluidic channel or channels MFC. These walls define a closed microfluidic channel MFC.
  • the microfluidic channel MFC may define a cavity having, for example, a substantially honeycomb or circular cross-sectional profile, or a substantially square or rectangular cross-sectional profile, as shown for example in Figure 4.
  • the microfluidic channel MFC has, for example, a height between 500nm and 500pm or between 5pm and 50pm (for example between the upper FL and lower CL walls FL), and/or a width between 500nm and 500pm or between 5pm and 50pm (for example, between side walls wl, w2), and/or a length between 5pm and 5cm or between 50pm and 2cm (for example, in the flow direction F (see Figure 4)).
  • separation walls may also be present to define a plurality of microfluidic channels in the unit 111 separated by these separation walls.
  • the plurality of microfluidic channels may comprise independent microfluidic channels in which, for example, a different multi-layered assembly MLA of biochemical species is present.
  • the plurality of microfluidic channels may alternatively or additionally be interconnected via a flow channel or lane fl (see for example Figure 3) that can be opened or closed to establish or not the interconnection using a controller or control channel.
  • the antibody unit 111 may alternatively comprise a plurality of elongated sections si containing solely one microfluidic channel extending therethrough and the plurality of sections may be interconnected using the previously mentioned flow channels fl ( Figure 3).
  • the walls include or define a surface configured to receive the multi-layered assembly MLA of biochemical species.
  • the walls may extend substantially parallel to each other.
  • the walls can define a curved surface delimiting the microfluidic channel MFC.
  • the walls define a concave curvature 304c as shown in Figure 3.
  • the microfluidic channel MFC can include an array or plurality of elongated structures MP (see for example Figure 3).
  • the elongated structure MP extends from a wall of the antibody unit 111 and inside the microfluidic channel MFC.
  • the elongated structure or structures MP may extend from a first wall towards a second wall and may contact the second wall.
  • the elongated structure M P may extend from the lower wall or floor FL towards the upper wall or ceiling CL and may contact the upper wall or ceiling CL.
  • the elongated structure or structures M P may extend from a first side wall wl towards the second side wall w2 and contact the second side wall w2.
  • the elongated structures extend from a surface of the microfluidic channel MFC and extend inside the microfluidic channel M FC.
  • the elongated structures may extend in a direction non-parallel or substantially perpendicular to a direction of fluid flow.
  • the elongated structures MP may also contact a wall and extend along the length of the wall. For example, contact and extend along the side wall wl and/or side wall w2 between the lower wall or floor FL towards the upper wall or ceiling CL.
  • the array of elongated structures is composed, for example, of a repeating pattern 304a, 304b of elongated structures.
  • the pattern can, for example, comprise a plurality of elongated structures disposed as a matrix or grid array.
  • the repeating pattern can extend partially or fully through the antibody unit 111.
  • the elongated structure MP is or defines, for example, a micropillar.
  • the or each elongated structure has, for example, a height between 5pm and 50pm (for example between the upper FL and lower CL walls FL), and/or a width between 5pm and 50pm (for example, in the X- direction (see Figure 3), and/or a length between 5pm and 50pm (for example, in the Y-direction (see Figure 3)).
  • the topology or arrangement of the elongated structure MP in microfluidic channels MFC is determined in order to obtain a high surface area, to miniaturize the overall device footprint and ensure a flawless or extremely high distribution of chemical species that is without or with minimized dead volumes where undesired chromatin could accumulate.
  • the elongated structure can have or define for example a circular cross-section, a square or rectangular cross section 304b, or preferably a rhomboidal cross section 304a, for example, with the major axis aligned to the direction of the flow.
  • the plurality of elongated structures MP may all have the same cross-sections or different or a mix of different cross-sections.
  • the plurality of elongated structures MP in the antibody unit 111 or each antibody unit 111 defines, for example, a surface area to volume ratio of between 0.5 micrometer 1 and 5 micrometer 1 .
  • the plurality of elongated structures MP in the microfluidic channel MFC or each microfluidic channel MFC defines a surface area to volume ratio of between 0.5 micrometer 1 and 5 micrometer 1 .
  • Figure 3 shows the plurality of elongated structures MP in the microfluidic channel MFC of a subpart 303 of the antibody unit 111.
  • the content of this subpart 303 is repeated extending through section si to define, for example, an immunoprecipitation (IP) lane (see also Figure 7a).
  • the antibody unit 111 of Figure 3 includes a plurality of IP lanes, for example, fives interconnected IP lanes.
  • the microfluidic device or chip 108 can include a plurality of antibody units 111 in which the plurality or each antibody unit 111 includes at least one or a plurality of interconnected IP lanes, as for example shown in Figure lb or Figure 7a.
  • microfluidic channel MFC defines or results in a plurality of interconnected microfluidic canals or passages MCA (see Figure 7) being present between adjacent elongated structures in the antibody unit 111 and through which a fluid can flow through.
  • the microfluidic canal or passage has, for example, a height between 5pm and 50pm (for example between the upper FL and lower CL walls FL), and/or a width between 5pm and 50pm (for example, in the X-direction ( Figure 3) between two elongated structures MP), and/or a channel length between 5pm and 50pm (for example, in the Y-direction ( Figure 3) between two elongated structures MP).
  • the microfluidic device 108 includes at most 7425 microfluidic canals or passages MCA, and at most 4400 elongated structures MP.
  • Each antibody unit includes at most 135 microfluidic canals or passages MCA, and at most 80 elongated structures MP.
  • Each elongated structure MP also delimits or includes a surface configured to receive a multi-layered assembly MLA of biochemical species for capturing a target molecule.
  • the multi-layered assembly MLA is provided on or attached to this surface of the elongated structure MP to provide a biofunctionalized surface for capturing a target molecule.
  • the surface of the walls defining the microfluidic channel MFC and/or the surface of the elongated structures MP comprise or consist of a hydrophobic surface or a hydrophilic surface.
  • the walls or the surface of the walls defining the microfluidic channel MFC and/or the elongated structures MP or the surface of the elongated structures MP include or consist solely of a polymer.
  • the polymer is, for example, configured to passively adsorbs an attachment layer or first layer 401 of the multi-layered assembly MLA.
  • the polymer can, for example, consist solely of or comprise polydimethylsiloxane (PDMS), or Polymethylmethacrylate (PMMA), or Cyclic-olefin copolymer (COC), or POLYSTYRENE (PS), or POLYCARBONATE (PC), or POLY-ETHYLENE GLYCOL DIACRYLATE (PEGDA), or POLYURETHANE (PU), or polyfluoropolyether diol methacrylate (PFPE-DMA), or TEFLON.
  • PDMS polydimethylsiloxane
  • PMMA Polymethylmethacrylate
  • COC Cyclic-olefin copolymer
  • PS POLYSTYRENE
  • PC POLYCARBONATE
  • PEGDA POLY-ETHYLENE GLYCOL DIACRYLATE
  • the surface of the walls defining the microfluidic channel MFC and/or the elongated structures MP is biofunctionalized and includes at least one antibody and/or at least one molecular capturing agent attached thereto.
  • the antibody is, for example, specific to a DNA binding protein.
  • the microfluidic channel MFC and the elongated structures MP include, attached to or provided on the hydrophobic or hydrophilic surface, a first layer 401 assembled by deposition of a first protein species to the hydrophobic or hydrophilic surface.
  • the first layer 401 is, for example, assembled by adsorption of a first protein species to the surface of the microfluidic channel MFC and/or the elongated structures MP.
  • the first protein species comprises or consist of, for example, biotinylated bovine serum albumin (biotinylated-BSA).
  • the multi-layered assembly MLA of biochemical species includes a second layer 402 composed of a second protein species having (high) biophysical affinity to the first protein species.
  • the second protein species comprises or consists of, for example, a protein of the avidin family or a glutaraldehyde.
  • the multi-layered assembly MLA can further include a third layer 403 composed or consisting of a third protein species with (high) biophysical affinity to the second layer or assembly 402, or with an antibody or biotinylated antibody.
  • the third protein species comprises or consists of a protein A or protein G or protein A/G, or a biotinylated protein A or biotinylated protein G or biotinylated protein A/G.
  • the multi-layered assembly MLA can further include a fourth layer 404 composed or consisting of an antibody or capturing agent.
  • the multi-layered assembly MLA can comprise or consist solely of the first layer 401, the second 402, the third 403, and the fourth 404 layer.
  • the first 401, second 402, third 403, and/or fourth 404 layers can be assembled by flowing a concentrated protein solution through the microfluidic channel or channels MFC of the antibody unit 111, 201.
  • Figure 4 schematically shows the multi-layered assembly MLA on one of the side walls w2.
  • the multi-layered assembly MLA can also be preferably present on the other side wall wl and the upper CL and lower FL walls as wells on the surfaces defines by the elongated structures MP.
  • the multi-layered assembly MLA is configured to target any molecule to which a specific antibody can bind.
  • the multi layered assembly MLA is, for example, configured to target protein/DNA complexes, transcription factors, histone modifications or DNA methylation.
  • a method for fabricating a system 1 for immunoprecipitation of a protein-DNA complex or for capture of a protein-DNA complex of the present disclosure may include providing the system 1 wherein the microfluidic channel or channels MFC and/or the elongated structures MP are non-biofunctionalized; and flowing a concentrated protein solution through the microfluidic channel or channels MFC of the antibody unit 111, 201 to assembly a plurality of layers 401, 402, 403, 403 on at least one surface of the microfluidic channel or channels MFC and/or the elongated structures MP to biofunctionalize the microfluidic channel or channels (MFC) and/or the elongated structures MP.
  • the multi-layered assembly MLA defines a multilayered "totem" of molecular species.
  • the totem is based on strong although non-covalent molecular interactions and culminates with the immobilization of an antibody of choice prior to immunoprecipitation.
  • the first layer can be, for example, obtained by flowing a concentrated solution of biotinylated-BSA into and through the microfluidic device or chip 108, which passively and thoroughly adsorbs to the
  • This layer has both an insulating role, that is, it prevents non-specific adsorption of chromatin, and a docking role for the next layer, which can be obtained by flowing a solution containing, for example, neutravidin that strongly binds to the biotin groups of the first layer.
  • the third layer can be formed by flowing a solution of, for example, biotinylated-protein A/G, which gets firmly immobilized by the unsaturated binding sites of the previous layer.
  • a recombinant protein having ability to strongly bind to a large number of different antibodies is thus preferably used.
  • the multi-layered assembly MLA is configured to retain this ability and constitutes a general substrate for antibody pull down.
  • Each antibody unit 111 can, for example, be configured to capture a different target molecule or to carry out a different immunoprecipitation reaction.
  • the system 1 further includes at least one or a plurality of inlets 107 for providing a sample containing, for example, DNA binding proteins to the microfluidic device 108.
  • the inlet 107 is configured for providing, for example, a cross-linked sheared or sonicated chromatin to the microfluidic device 108.
  • the inlets 107 are in fluid communication with the microfluidic device 108 and the microfluidic device 108 includes openings for fluid communication with the inlets 107.
  • the inlets 107 are configured to be open or closed to allow or prevent fluid communication with the microfluidic device 108. This is done, for example, using control valves 104 actioned via a microcontroller 102 and computer/processor 101 using a system control program or software SW. This allows to select individual anti-body unit or units 111, or individual IP lane or lanes for sample input. Alternatively, all anti-body units 111, or IP lanes can be selected via use of a common inlet 204 (see for example Figure 2).
  • the system 1 also includes inlet collection tubes 106 in fluid communication with the inlets 107.
  • the inlet collection tubes 106 are configured to provide input elements to the microfluidic device 108.
  • the inlet collection tubes 106 are configured to hold and release samples to the microfluidic device 108 to be processed by the microfluidic device 108.
  • the inlet collection tubes 106 are also used to form the multi layered assembly MLA in the microfluidic device 108.
  • the contents of one inlet collection tubes 106 can, for example, be provided to all anti-body units 111 of the microfluidic device 108.
  • the system 1 also includes one or a plurality of outlets 110 for providing DNA or eluted DNA, from the microfluidic device 108, for sequencing.
  • the outlets 110 are connected to the microfluidic device 108 in a same manner as that of the inlets 107 and are controlled in the same manner.
  • the system 1 can further include outlet collection tubes 109 connected to the microfluidic device 108 outlets 110 for collecting processed samples from the microfluidic device 108.
  • the collecting processed samples from the microfluidic device 108 can also be re-inserted by additional inlets into the microfluidic device 108 for further processing, for example, by one or more different anti body units 111. This, for example, allows sequential ChIP-seq to be performed.
  • the system 1 can further include a calculator or calculating means 101, a microcontroller unit 102 and a valve device comprising a plurality of electromechanical (EM) valves 104.
  • EM electromechanical
  • a temperature control device for controlling the temperature of system elements such as the microfluidic device 108 may also be optionally included.
  • a PCR device for controlling the temperature of system elements such as the microfluidic device 108 may also be optionally included.
  • the calculator or calculating means 101 can be, for example, a general-purpose computing unit 101 used for experiment scripting.
  • the system control program or software SW executed by the calculator or calculating means 101 allows operation of the system 1 to be carried out and the methods of the present disclosure to be performed.
  • the microcontroller unit 102 is connected to the computer 101 and to an arbitrary number or plurality of electromechanical (EM) valves 104. This unit 102 translates the computer instructions into voltage signals. It is connected to the EM valves by means of electric wires 103.
  • EM electromechanical
  • the arbitrary number or plurality of EM valves 104 are connected to the microcontroller unit 102 and to the set of collection tubes 106 which, for example, contain experimental samples or other solutions to be inputted to the microfluidic device or chip 108.
  • EM valves 104 and collection tubes 106 are, for example, connected through airtight connections 105 in the form of plastic (or rubber) tubing (or wells).
  • the EM valves 104 are configured to allow or block the passage of pressurized air to the microfluidic device or chip 108.
  • the microfluidic chip 108 contains an arbitrary number or plurality of antibody (or IP) units 111, 201.
  • the microfluidic chip or device 108 is connected to the inlet collection tubes 106 and the outlet connection tubes 109 through airtight connections 110 in the form of, for example, plastic (or rubber) tubing (or wells).
  • the valve device comprising the plurality of electromechanical (EM) valves 104 is configured to receive a pressurized fluid, for example, air and configured to distribute the pressurized air to multiple outlets of the valve device (see for example, Figure lib).
  • the outlets each comprise an EM valve 104 that is controlled via the microcontroller unit 102.
  • the air pressure value at the outlet can also be controlled and set to a desired value.
  • the plurality of EM valves 104 are connected, via the inlet collection tubes 106, to flow lines LF and control lines LC of the microfluidic chip or device 108 (see for example, Figure lib).
  • the plurality of EM valves 104 are operated to allow or block the passage of a pressurized fluid (provided for example via the inlet collection tubes 106) to the microfluidic device 108.
  • Valves 104 connected to flow lines LF permit to distribute a fluid through flow lines and compartments such as the microfluidic channels MFC to allow, for example, the immunoprecipitation reaction to be carried out inside the antibody units 111 of microfluidic chip 108.
  • Valves 104 connected to a control line LC permit to select the direction or destination of the fluid flow in the microfluidic device 108.
  • these valves 104 connected to a control line LC permit to select which antibody units 111 fluid is flown to and are thus used in the microfluidic device 108, or the sequence of antibody units 111 a fluid is flow through. This allows different biofunctionalisation of the antibody units 111 and also to perform multiplexed measurements or processing of samples in the microfluidic device (108).
  • the microfluidic device 108 can, for example, comprise a superposed control layer and flow layer in which are defined superposed control lines and flow lines separated by an intermediate blade or wall. Fluid pressure applied to the control line deforms the intermediate blade to allow opening and closing of the flow line to control fluid flow in the flow line.
  • valves 104 are activated via the microcontroller unit 102 and the calculating means 101 with the desired processing in the microfluidic device (108) being determined via the experiment scripting or the system control program SW executed by the calculating means 101.
  • the actual immunoprecipitation reaction is carried out inside of the FloChIP microfluidic chip 108 (see for example, Fig. 2).
  • the device 108 itself is composed of an arbitrary number or plurality of antibody units 201, one for each immunoprecipitation reaction.
  • This array of units can be connected to the collection tubes 106 either in parallel through the common inlet 204 and common outlet 206, or individually through the individual inlets (202, one inlet per unit) and individual outlets (205, one outlet per unit).
  • important elements of the FloChIP approach are combined. These elements are: the antibody unit (Fig. 3) and an innovative surface biofunctionalisation (Fig- 4).
  • Each antibody unit 111 can be composed of a large number of serially connected microfluidic microstructured units. Examples of these microstructured units 304a, 304b are reported in a close-up view of a subpart 303 of the antibody unit 111.
  • the microstructures MP have the role of increasing the surface area-to-volume ratio of the antibody unit 111.
  • These microstructures MP can be implemented for example in the form of micropillars with different cross-sectional shapes.
  • the cross-section of the micropillars can be rhomboidal 304a, squared 304b, or circular.
  • the micropillars are not essential for the basic functioning of the above described biofunctionalization chemistry and a unit without pillars 304c also functions. However, best results are obtained in the presence of micropillars.
  • the use of fluidic channels (or passages) and microstructures with microscopic dimensions is also important. Therefore, microfluidic channels (or passages) and microstructures are preferred to have heights ranging between 5 and 50 micrometers.
  • the surface biofunctionalisation is represented by the multilayered assemble MLA of biochemical species (Fig. 4, 7a, 11a).
  • the function of this surface biofunctionalisation is accommodating a wide variety of antibodies and other molecular capturing agents in order.
  • the first layer 401 of FloChIP surface biofunctionalisation is assembled by passive adsorption of a protein species to the walls of the microfluidic channels. For this reason, any polymer that supports passive adsorption of protein species can be used for FloChIP.
  • the polymer used is for example polydimethylsiloxane (PDMS) but any hydrophobic or hydrophilic polymer can be used as a viable substrate and the protein species used for this first layer can be, for example, biotinylated-BSA.
  • the second layer 402 is composed of a protein species with high biophysical affinity to the protein species of the first layer 401.
  • this protein can be, for example, a protein of the avidin family.
  • the third layer 403 is also composed of a protein species with high biophysical affinity to the second layer 402.
  • this protein can be, for example, biotinylated protein A or protein G or protein A/G.
  • the fourth layer 404 is a layer composed of the antibody or capturing agent of choice.
  • All the layers mentioned above are assembled, for example, by flowing a concentrated protein solution through the microfluidic channels of the antibody unit 111.
  • Alternative multilayered assemblies could be formed by using, for example, the following sequence of protein/chemical solutions:
  • Layer 1 biotinylated BSA
  • layer 2 avidin
  • layer 3 biotinylated antibody.
  • Layer 1 BSA; layer 2: glutaraldehyde; layer 3: protein A or G or A/G, layer 4: antibody.
  • Layer 1 BSA
  • layer 2 glutaraldehyde
  • layer 3 antibody.
  • the multilayered surface biofunctionalisation is ready to be used to capture the target molecules.
  • the possible target molecules are any molecule to which a specific antibody can bind. Examples include, but are not limited to, transcription factors, histone modifications and DNA methylation.
  • the present disclosure also concerns a method for immunoprecipitation of a protein-DNA complex or for capture of a protein-DNA complex.
  • the method includes the steps of providing the system 1 of the present disclosure, providing at least one sample containing DNA binding proteins to the microfluidic device 108, and recuperating, at the least one or a plurality of outlets of the microfluidic device 108, DNA or eluted DNA for sequencing.
  • the method is, for example, a chromatin immunoprecipitation method.
  • the providing step can include, for example, providing a cross-linked sheared or sonicated chromatin to the microfluidic device 108.
  • the method may further include the step of preparing the DNA for sequencing by providing a transposase loaded with NGS adapters in the microfluidic chip 108 for attachment to the DNA; and eluting the DNA in the microfluidic chip 108.
  • Eluting the DNA in the microfluidic chip 108 can be carried out by applying thermal energy and a salt buffer.
  • the method may further include the step of carrying out sequencing of the DNA.
  • the sequencing can be carried out, for example, using next generation sequencing (NGS) processing.
  • NGS next generation sequencing
  • the method may further include the step of carrying out on-chip library indexing to decrease downstream library preparation time.
  • Carrying out on-chip library indexing to decrease downstream library preparation time can, for example, comprise flowing, in the microfluidic device 108, a transposase loaded with NGS adapters on top of the immunoprecipitated chromatin.
  • the provision of a sheared or sonicated chromatin to the microfluidic device 108 may include providing samples containing less than 1000, or less than 500 cells.
  • the present disclosure also concerns an apparatus configured to carry out the above described method.
  • protein/DNA complexes are immunoprecipitated using FloChIP and subsequently the DNA is prepared for next generation sequencing (NGS) directly on chip.
  • NGS next generation sequencing
  • This preparation is, for example, carried out by flowing in the microfluidic chip 108 a transposase loaded with NGS adapters on top of the immunoprecipitated chromatin.
  • This is a variant of a patented technology called “ChIPmentation” [12, P2]
  • the DNA is then eluted with high temperature and high salt buffer in order to be processed by next generation sequencing (NGS).
  • NGS next generation sequencing
  • FloChIP-derived data is very consistent with the benchmark data. Moreover, the proportion of mapped reads that fall within ChIP-seq peaks tend to be the same - if not higher - for FloChIP as compared to ENCODE (Fig. 5f), thus corroborating the efficiency of the system and method of the present disclosure. Finally, as observed in Fig. 5g, the superior inter-experiment correlation between two FI3k27ac replicates advocates for the high reproducibility of the system and method of the present disclosure. It is noteworthy that, ENCODE experiments were performed with an average of 10 million cells for each ChIP, whereas FloChIP was performed with only 100 thousand cells. This demonstrates that not only can FloChIP achieve the comparable genomic coverage as ENCODE, but it can do so with substantially less cellular input.
  • the lower limits of detection of the system and method of the present disclosure was also investigated.
  • the inventors performed a multiplexed experiment in which was used the same antibody target (i.e. FI327ac in this case) for seven different samples of decreasing cellular input, from 400,000 cells to as few as 500 cells.
  • the inventors obtained high enrichment for all input dilutions, therefore proving that the system and method of the present disclosure can selectively enrich the target sites for a very low number of cells.
  • the inventors proceeded to sequence the two samples at the extremes of our dilutions (i.e. with 500 and 400,000 cells) and then evaluate the correlation.
  • FloChIP is a preferable solution for user-friendly and reliable ChIP-seq experiments.
  • the microfluidic device comprises or consists, for example, of a flow and a control layer. Molds for each layer can be fabricated using standard lithography techniques on 4" silicon wafers. After exposure to 02 plasma the control layer mold is patterned with SU- 8 photoresist to a height of, for example, 10 pm. The mold for the fluidic layer was fabricated with AZ9260 photoresist to a height of, for example, 20 pm. Devices were cast in polydimethylsiloxane (PDMS), for example, using multilayer soft lithography. PDMS was prepared at a 20:1 ratio and spin-coated on the flow layer mold at 1700 rpm.
  • PDMS polydimethylsiloxane
  • PDMS at a 5:1 ratio was cast on the control layer mold to a thickness of about 4 mm. Both layers were baked at 80 °C for 30 min. The control layer was peeled off from its mold and manually aligned to the flow layer mold, followed by a baking step at 80 °C for 90 min.
  • control lines on the microfluidic device 108 were primed with phosphate buffered saline (PBS) at 5 psi.
  • PBS phosphate buffered saline
  • the pressure was increased to 25 psi.
  • Flow lines were operated at 3 psi.
  • the surface area was derivatized by flowing, for example, a solution of biotinylated BSA, followed by a 1 min wash in PBS.
  • a neutravidin solution in PBS was flown, followed by a 1 min wash in PBS.
  • a biotinylated protein A/G in PBS was flown, followed by a 1 min wash in PBS.
  • GM12878 cells samples were centrifuged at l,600g for 5 min at room temperature in a swing bucket centrifuge with soft deceleration. Cells were then washed twice with PBS at room temperature by centrifugation and resuspension. Cells were cross-linked for 5 min with 1 ml of 1% freshly prepared formaldehyde. Cross-linking was terminated by adding 0.05 ml of 2.5 M glycine and shaking for 5 min at room temperature. Cross-linked cells were pelleted and washed with precooled PBS buffer and resuspended in sonication buffer. Cross-linked cells were sonicated with a Covaris E220 sonicator.
  • the sonicated lysate was centrifuged at 14,000g for 10 min at 4 °C. Sonicated chromatin in the supernatant was transferred to a new 1.5-ml LoBind Eppendorf tube. From this stock chromatin preparation, samples equivalent to 400 ⁇ 00, 100 ⁇ 00 and 500 cells were divided into aliquots and diluted to give a final volume of 50 pi.
  • the FloChIP system 1 is engineered for automated, bead-less and miniaturized ChIP-seq.
  • the two core elements of FloChIP's technology are the assembly of a multilayered "totem" of molecular species (Fig. 7a, Supp. Fig. 11a) and an engineered pattern of high surface-to-volume micropillars (Fig. 7b).
  • the totem is based on strong although non-covalent molecular interactions and culminates with the immobilization of an antibody of choice prior to immunoprecipitation.
  • the first layer is obtained, for example, by flowing on-chip a concentrated solution of biotinylated-BSA, which passively but thoroughly adsorbs to the hydrophobic walls of the microfluidic device 108. This layer has both an insulating role, i.e.
  • the third layer is formed, for example, by flowing a solution of biotinylated- protein A/G, which gets firmly immobilized by the unsaturated binding sites of the neutravidin layer.
  • Protein A/G is a recombinant protein used in a variety of immunoassays due to its ability to strongly bind to a large number of different antibodies. This ability is retained by FloChIP's totem which thus constitutes a general substrate for antibody pull-down (Fig. 7a).
  • the only substrate requirement is the hydrophobic or hydrophilic surface, for example, a hydrophobic surface of the polymer. Therefore, the inventors set out to optimize the topology of the microfluidic channels having three main goals in mind: obtaining as much surface area as possible, miniaturizing the overall device footprint and ensuring flawless distribution of chemical species - i.e. without dead volumes where undesired chromatin could accumulate.
  • This optimization strategy led to a preferred design encompassing an array of micropillars of rhomboidal cross-section with the major axis aligned to the direction of the flow (Fig. 17b).
  • the micropillar pattern is repeated multiple times across each IP-lane (Fig. 7c).
  • the inventors first sought IP chromatin derived from a FleLa FI2B-mCherry cell line using an anti-mCherry antibody.
  • the resulting fluorescence micrographs confirmed the role of each layer of the totem for successful IP of cellular chromatin (Fig. 7d).
  • the IP-lane is an important element of the FloChIP architecture and it can itself be repeated n times, where n is the desired throughput of the device.
  • n is the desired throughput of the device.
  • a network of valves for example, Quake-style microfluidic valves was used in the system 1.
  • different multiplexing modes can be achieved with the same microfluidic architecture.
  • FloChIP mode 1 provides the option of multiplexing one sample into different IP units, hence equally distributing the same sample across multiple lanes, enabling multiple parallel IPs involving distinct antibodies (Fig. 8a).
  • FloChIP mode 2 provides the option of coating the whole device with one antibody, thus achieving sample multiplexing (Fig. 8b).
  • both multiplexing modes are compatible with the direct chromatin immunoprecipitation ChIP approach.
  • FloChIP is also fully compatible with the indirect ChIP strategy, in which the chromatin is pre-incubated with an antibody before flowing the sample-antibody mixture on-chip.
  • the FloChIP system 1 reliably reproduces ENCODE data across a wide range of input cells.
  • the inventors first set out to empirically estimate the overall binding capacity of each IP lane.
  • the inventors performed FloChIP in multiplexing mode 2, i.e. "antibody multiplex”, by functionalizing the whole chip with an anti-FI3K27ac antibody and immunoprecipitating different chromatin dilutions, from 1 million down to 500 cells.
  • FloChIP's derived libraries for four dilutions, i.e. 100 ⁇ 00, 50 ⁇ 00, 5 ⁇ 00 and 500 cells. Although the rate of uniquely mapped reads remained high for all samples (Fig. 12c), the fraction of reads falling into peaks (FRiP score) slightly decreased with lowering input amounts - from over 60% for 100 ⁇ 00 cells, to just above 10% for 500 cells (Fig. 12d).
  • genome-wide analysis of the obtained libraries revealed expected accumulation of reads into regions in proximity of transcription start sites (TSS, Fig. 8f).
  • the system and method of the present disclosure advantageously allows immunoprecipitation from 10 s cells to as low as 500 cells as well as immunoprecipitation of different histone marks.
  • the inventors obtain data with quality comparable to the benchmark ENCODE data with less cells and, in shorter times, in an automated and parallelized way. Thus, a system of high sensitivity, efficiency and multiplexing is assured.
  • the FloChIP "sequential-IP" mode or method of the system 1 of the present disclosure additionally provides genome-wide information on bivalent promoters.
  • Conventional ChIP-seq provides information on the genome-wide localization of one specific protein or histone modification at a time.
  • DNA regulatory elements generally harbor the interaction of several transcription factors and histone modifications in order to regulate gene expression. For instance, it has been shown that promoters showing both repressive (FI3k27me3) and activating (FI3k4me3) marks are a characteristic feature in embryonic stem (ES) cells.
  • This class of promoters have been originally named "bivalent" and are strongly associated to key developmental genes.
  • sequential-ChIP was developed.
  • Sequential-ChIP relies on the consecutive IP of two different antigens and, as opposed to simply intersecting two ChIP-seq datasets, provides unbiased information on bivalent regions. Despite the advantage of sequential ChIP over standard ChIP in discerning true bivalency, its manual involvement and impracticality have thus far prevented widespread usage. Moreover, due to the inefficiency of the method, few studies have so far performed sequential ChIP followed by next generation sequencing (sequential-ChIP-seq), since most of them relied on qPCR to validate putative bivalent regions (sequential ChIP-qPCR).
  • the inventors exploited FloChIP's intrinsic modularity, highly efficient IP and multiplexing features to derive the example of an automated and miniaturized sequential-ChIP solution (Fig. 9a).
  • the inventors validated their method by focusing on bivalent chromatin given its well studied role in embryonic development. Specifically, the inventors acquired genome-wide direct co-occupancy profiles for H3K27me3 and H3K4me3 in mouse embryonic stem cells (mESCs) in both IP directions - i.e. H3K27me3 first followed by H3K4me3 (H3K27me3/H3K4me3) and vice versa.
  • mESCs mouse embryonic stem cells
  • H3K4me3 and H3K27me3 bivalency has been originally attributed to promoters of developmental genes, leading to the hypothesis that a bivalent state maintains genes in a poised state.
  • promoters show three distinct patterns of bivalency, i.e. pseudo bivalency, partial bivalency and full bivalency.
  • pseudo bivalency i.e. pseudo bivalency
  • partial bivalency i.e. partial bivalency
  • full bivalency i.e. pseudo bivalency
  • the inventors consider these classes an artificial construct that does reflect the more fine-grained distribution of bivalency levels. Therefore, instead of assigning promoters to specific classes, the inventors compute for each TSS a "bivalency score" (bvScore, Fig. 9c, detailed further below).
  • HPC high-CpG
  • the system and method of the present disclosure advantageously allows sequential ChIP-seq on histone marks.
  • the obtained data shows a quality comparable to benchmark previously published data with less cells and, shorter times, in an automated and parallelized way and a system of high sensitivity, efficiency and multiplexing is provided.
  • the FloChIP system 1 is capable of ChIPing TFs in a "high-throughput" mode.
  • Previous attempts at improving the sensitivity and multiplexing ability of ChIP-seq experiments were shown to perform well only in the context of histone modifications. The reason for this is that performing TF ChIP-seq poses additional challenges as compared to histone marks (FIM) including the fact that i) TF/DNA interactions are less abundant and less robust than HM/DNA interactions and ii) antibodies for TFs normally show lower affinity for their epitopes as compared to HM antibodies.
  • FIM histone marks
  • the inventors After establishing a working protocol for TF ChIP-seq, the inventors set out to concurrently demonstrate the high-throughput capabilities of the system 1. To this end, by using half of the 64 IP lanes of the FloChIP device, the inventors performed MEF2-A ChIP-seq on chromatin derived from 32 different lymphoblastoid cell lines (LCLs) (Fig. 10a, A map of human genome variation from population-scale sequencing.)
  • ChIP-seq allows to probe DNA-protein interactions on a genome-wide scale, thus achieving high-throughput in terms of DNA sequence space coverage.
  • ChIP-seq remains at the lowest level possible, with only one protein species and one sample tested per experiment. Aggravating this aspect, the long and manually intensive protocol prevents straightforward development towards higher throughput.
  • Community-led efforts like ENCODE have therefore been put in place in order to perform ChIP-seq for a large number of proteins and cell types. However, despite the valuable data generated, ENCODE still sampled only a small portion of a much larger combination space.
  • ChIP-seq In addition to limited throughput and manual involvement, standard ChIP-seq is also restricted by the input requirements for biological material. The requirement for at least one million cells, has precluded ChIP-seq from performing reliably on smaller but possibly biologically relevant samples. Understanding the impact of these limitations, several groups attempted to improve the original protocol. However, these attempts have addressed specifically certain issues while overlooking others.
  • the system and method of the present disclosure address all major ChIP-seq limitations by introducing a new technology, FloChIP, that allows for rapid, high-throughput, automated and sensitive chromatin immunoprecipitation.
  • the two core technological aspects of FloChIP are its surface chemistry and its microfluidic architecture. The former confers FloChIP the ability to perform solid-state bead-less IP with most off-the-shelf antibodies, while the latter provides the structural substrate for miniaturized IP, rapid washing, multiplexing and straightforward automation.
  • the inventors performed FloChIP for a variety of targets and samples. Initially, the inventors aimed to empirically gain insights into FloChIP's dynamic input range. By obtaining high H3k27ac qPCR fold enrichment and high correlation with the respective ENCODE data for inputs ranging between 10 s and 500 cells, the inventors show that FloChIP can be used across a wide range of inputs.
  • the inventors show that the chromatin eluted after the first IP step can be re-directed into a second IP lane, therefore achieving straightforward sequential immunoprecipitation.
  • the inventors validated FloChIP's sequential IP by recapitulating previously published qPCR and sequencing data on bivalent promoters in mouse embryonic stem cells. To the best of the inventors knowledge, this is the fastest (1/2 day) and most sensitive (100 ⁇ 00 cells) example of sequential ChIP-seq. Moreover, this is the first automated, microfluidic and bead-less example of sequential ChIP-seq.
  • the FloChIP system and method of the present disclosure are a robust, sensitive and high-throughput all-in-one ChIP-seq solution. Given its advantages and wide applicability, the FloChIP system 1 can be a widely adopted tool for the study of genome-wide protein-DNA interactions.
  • GM 12878 cells (5-10 millions) were harvested, washed once with PBS and resuspended in 1ml crosslinking buffer (1% PFA in PBS) for 10 minutes shaking. Crosslinking was stopped by adding 50mI of 2.5M glycine and shaking for other 5 minutes. Fixed cells were then washed twice with ice-cold PBS, pelleted, deprived of the supernatant, snap frozen and stored and -80°C.
  • the frozen cell pellet was resuspended in ice-cold PBS at 4°C agitating for 30 minutes, spun at lOOOg for 5 minutes, resuspended in lysis buffer (50 mM Flepes pH 7.8, 140 mM NaCI, ImM EDTA, 0.5% NP40, 10% glycerol, 0.25% Triton and freshly added protease inhibitor), incubated with mild agitation for 10 minutes, spun for 5 minutes at lOOOg, resuspended in nuclei wash buffer (20 mM T ris-HCI pH 8.0, 200 mM NaCI, 1 mM EDTA, 0.5 mM EGTA and freshly added protease inhibitor), incubated with mild agitation for 10 minutes, spun for 5 minutes at lOOOg and resuspended in sonication buffer (20 mM T ris-HCI pH 8.0, 200 mM NaCI, 1 mM EDTA, 0.5
  • Nuclei were sonicated on a covaris E220 machine with the following settings: MOW intensity, 5% duty factor and 200 bursts/cycle. Chromatin was then aliquoted (" ⁇ 00 ⁇ 00 cells/aliquot) in PCR tubes and snap frozen until ChIP.
  • microfluidic device designs were generated using Tanner L-Edit and fabricated using multilayer standard soft lithography (Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science 298, 580-584, 2002) at the EPFL center for microtechnology. Briefly, designs were first transferred to chrome masks using a VPG200 pattern generator (Heidelberg Instruments). Subsequently, microfluidic molds were assembled on silicon wafers with SU8 photoresist for the control layer and AZ9260 positive resist for the flow layer using a SUSS ACS200 Gen3 system (SUSS MicroTec).
  • Microfluidics chips were fabricated by first separately casting PDMS onto the SU8 and the AZ9260 wafers with two different PDMS/curing agent ratios (20:1 and 5:1, respectively), partially curing for 30 minutes at 80°C, peeling off the PDMS from the AZ9260 wafer and aligning it to the SU8 wafer in order the reconstitute the wanted pattern. The chips were finally fully cured at 80°C for one hour and half, peeled off, holed and plasma-bonded to clean glass slides or to PDMS- coated petri dishes.
  • Automated control of the FloChIP experimental workflow is obtained by a system of exemplary components including: 1) MATLAB software SW, 2) a standard laptop 101, 3) a WAGO fieldbus controller (ModBus 750-881) 102, 4) FESTO 3/2 way 24V miniature solenoid valves 104, 5) compressed air building supply (Fig lib) and 6) a PCR machine. Tygon tubing and western blot tips are used to interface the microfluidic chip 108 and the solenoid valves.
  • FloChIP is, in essence, a method consisting of the sequential introduction of different reagents into a custom-designed microfluidic chip 108.
  • This sequence of reagents can be programmed with simple scripting commands which are, in turn, translated into sequences of solenoid valve actuations and releases.
  • the concerted action of the solenoid valves, belonging to both the control layer and the flow layer of the chip 108, realizes in an automated fashion the required surface chemistry, immunoprecipitation and tagmentation reactions.
  • On-chip temperature control can be achieved by placing the microfluidic device 108 on top of a PCR machine with flat heat-block and starting a pre-programmed temperature sequence in sync with the MATLAB script.
  • a FloChIP method or experiment starts by pre-loading the control lines with distilled water and activating all valves (at a pressure of 25-30 PSI for the control lines and 2.5-5 PSI for the flow valves). Subsequently, all the reagents for the surface chemistry (i.e. biotin-BSA, neutravidin, PBS and biotin-protein A/G, antibodies), IP (chromatin), washes (low-salt, high-salt and LiCL buffers), tagmentation (Tn5 buffer) and elution (SDS buffer), are loaded into pipette tips and inserted into the inlets of the microfluidic device 108. At this stage, all valves are closed and there is no possible cross-talk between any of the reagents above.
  • all the reagents for the surface chemistry i.e. biotin-BSA, neutravidin, PBS and biotin-protein A/G, antibodies
  • IP chromatin
  • washes low-salt, high
  • the automated protocol is launched by running the respective script.
  • the exemplary protocol entails, in sequential order, the following steps: 20 minutes of BSA-biotin (2mg/ml), 30 seconds of PBS wash, 20 minutes of Neutravidin (lmg/ml), 30 seconds of PBS wash, 20 minutes of biotin-protein A/G (2mg/ml) and 30 seconds of PBS wash.
  • immunoprecipitation is carried out in two different ways: loading the chromatin mix into the IP units pre-functionalized with antibodies (direct ChIP) or loading of the pre incubated antibody/chromatin mix (indirect ChIP).
  • the antibody and chromatin are incubated for 2 or 4 hours in a PCR tube prior the loading on-chip.
  • the antibody/chromatin mixes are loaded into the chip in separate IP units by utilizing the same ON/OFF cycles as mentioned above.
  • the overall immunoprecipitation is performed at room temperature time spans between 30 and 60 minutes, depending on the amount of chromatin mix to be processed.
  • Tn5 buffer (10 mM Tris pH 8.0, 5 mM MgCh) is slowly flown on-chip at 37°C for 45 minutes. This step ensures the complete tagmentation of the immunoprecipitated chromatin.
  • SDS buffer (10 mM Tris pH 8.0, 200 mM NaCI, ImM EDTA, 1% SDS) is loaded on-chip at 65°C for 10 minutes in order to elute the antibody-bound chromatin from the device. The eluate is independently collected from each IP lane into PCR tubes and decrosslinked at 65°C for 4 hours. Following decrosslinking, DNA is purified in Qiagen EB buffer using Qiagen MinElute purification kits.
  • elution is performed by saturating the antibody with a given elution peptide (abl342 for H3k4me3, abl782 for H3k27me3 and ab24404 for H3k27ac, Abeam - Peptide elution buffer: 20mI of IP buffer, 2pg of an antibody-specific peptide).
  • a given elution peptide abl342 for H3k4me3, abl782 for H3k27me3 and ab24404 for H3k27ac
  • Abeam - Peptide elution buffer 20mI of IP buffer, 2pg of an antibody-specific peptide.
  • the chromatin is re-flown on-chip for the second immunprecipitation (Fig 9).
  • This second immunprecipitation is also performed using ON/OFF cycles of 2 minutes each.
  • the total time for the second ChIP is also between 30 and 60 minutes.
  • ChIP-qPCR Following FloChIP, qPCR was used to evaluate IP efficiency prior to next generation sequencing. qPCR was performed on a StepOnePlusTM (primer sequences: H3k27ac_FW CCACCCTGCACTT ACG ATG, H3k27ac_RV TGAGCTCCCTGTCTCTCCTC, H3k4me3_FW
  • Each qPCR reaction was composed of 10mI Applied BiosystemsTM PowerUpTM SYBRTM Green Master Mix, 0.8mI of a 10mM forward primer solution, 0.8mI of a 10mM reverse primer solution, 2mI of DNA and water to a final volume of 20mI.
  • the cycling program was the following: 2 minutes at 50°C, 2 minutes at 95°C and [5 seconds at 95°C, 20 seconds at 60°C]x60 cycles.
  • Fold enrichment values were obtained as ratios between the percent of input of the expected positive and negative regions genomic regions.
  • NGS Library preparation NGS library were prepared by mixing 20mI of purified DNA with 2.5mI of forward Nextera adapter, 2.5mI of reverse Nextera adapter, 32.5mI of NebNext master mix, 0.5mI of lx SYBR green and water to 65mI. First, 5 pre-amplification cycles are run as follows: 5 minutes at 72°C, 30 seconds at 98°C and [10 seconds at 98°C, 30 seconds at 63°C, 60 seconds at 72°C]x5 cycles.
  • DNA was size selected using AMPure XP beads in order to obtain a size distribution between 150bp and 500bp. Concentrations were measure with Qubit (ThermoFisher), size distribution was profiled with Fragment analyzer (AATI) and libraries were sequenced on an lllumina NextSeq 500.
  • Qubit ThermoFisher
  • AATI Fragment analyzer
  • Sequencing reads were mapped to the human (hg38 and hgl9) and mouse (mmlO) genomes using STAR (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013)) with default parameters.
  • Uniquely mapped reads were used to call peaks using FIOMER (Fleinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576-589 (2010)) command findPeaks.pl with the appropriate flag, i.e. -histone for histone marks and -factor for transcription factors.
  • FRiP scores we calculated using FIOMER's command annotatePeaks.pl and divided the total number of reads that fall within peaks by the total number of mapped reads. Correlation plots were generated using annotatePeaks.pl on a common peak file, either Encode's peak files or, alternatively, the overlapping set of peaks between Encode and FloChIP datasets.
  • the bvScore is assigned to each promoter and is intended to take into account both the intersection between two ChIP-seq datasets as well as the agreement between the respective sequential-ChIP-seq datasets. Accordingly, the bvScore can be expressed as the product of the co-occurrence score (cScore), which measures the relative coverage of the two ChIP-seq tracks, and the agreement score (aScore), which measures the relative coverage of the two sequential-ChIP-seq tracks.
  • cScore co-occurrence score
  • AScore agreement score
  • cScore (nmr4i+nmr27i)/(nmr4i-nmr27i), where nmr 4 , and nmr27i are the normalized number of mapped reads in promoter / for FI3K4me3 and FI3K27me3, respectively.
  • a positive cScore indicates prevalence of FI3K4me3 while a negative cScore indicates prevalence of FI3K27me3.
  • aScore the absolute value of (nmr 4 / 27j +nmr 27 / 4i )/(nmr 4 / 27 rnmr 27 / 4i ), where nmr 4 / 27i and nmr 27 / 4 , ⁇ are the number of mapped reads in promoter / for the two sequential-ChIP-seq experiments.
  • van Galen, P. et al. "A multiplexed system for quantitative comparisons of chromatin landscapes”.
  • Van Galen, P. et ai A multiplexed system for quantitative comparisons of chromatin landscapes. Mol. Cell 61, 170-180 (2015).

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Abstract

La présente invention concerne un système d'immunoprécipitation d'un complexe protéine-ADN ou de capture d'un complexe protéine-ADN comprenant : - un dispositif microfluidique comprenant une pluralité d'unités d'anticorps, chaque unité d'anticorps comprenant au moins un canal microfluidique, lesdits canaux microfluidiques comprenant au moins une surface conçue pour recevoir un ensemble multicouche d'espèces biochimiques permettant de capturer une molécule cible de façon à former une surface biofonctionnalisée ; au moins une entrée permettant de fournir au moins un échantillon contenant des protéines de liaison à l'ADN au dispositif microfluidique ; et au moins une sortie permettant de fournir de l'ADN ou de l'ADN élué destiné à un séquençage.
PCT/IB2018/060023 2017-12-13 2018-12-13 Système de capture ou d'immunoprécipitation d'un complexe protéine-adn Ceased WO2019116306A1 (fr)

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