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WO2021128034A1 - Procédé de séquençage haut débit pour l'accessibilité de chromatines monocellulaires - Google Patents

Procédé de séquençage haut débit pour l'accessibilité de chromatines monocellulaires Download PDF

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Publication number
WO2021128034A1
WO2021128034A1 PCT/CN2019/128154 CN2019128154W WO2021128034A1 WO 2021128034 A1 WO2021128034 A1 WO 2021128034A1 CN 2019128154 W CN2019128154 W CN 2019128154W WO 2021128034 A1 WO2021128034 A1 WO 2021128034A1
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cell
nucleus
water
sequencing
suspension
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Chinese (zh)
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张惠丹
赵星
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Suzhou Geno Truth Biotechnology Co Ltd
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Suzhou Geno Truth Biotechnology Co Ltd
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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

  • This application relates to a gene sequencing method, in particular to a novel high-throughput single-cell chromatin accessibility sequencing method, which belongs to the field of molecular biology.
  • Single-cell chromatin accessibility sequencing technology can detect the heterogeneity of single cells from the aspect of epigenetics, and provide accurate information for the diagnosis and treatment of diseases. Constrained by the throughput and cost of single-cell sequencing technology, many large-scale single-cell epigenetics research work cannot be carried out.
  • the traditional single-cell separation technology uses capillary tubes to separate single cells, which requires manual operation under a microscope, which has low throughput, time-consuming, and cumbersome process.
  • Another common method is to use flow cytometry to separate single cells. This method requires a large amount of samples, requires precise control, damages the cells, and requires high requirements for subsequent library building.
  • the Microwell-split pool system can also realize the separation of single cells, and its throughput is also low, usually 1000-5000 cells, the reaction volume is large, the reagent consumption is high, the cost is high, and the cell loss is large.
  • the 10X genomics and Biorad systems based on the principle of droplet microfluidics have higher throughput and can analyze the chromatin accessibility of tens of thousands of cells at the same time.
  • the 10X genomics and Biorad systems use microfluidic chips to wrap labeled beads and single cells in a droplet to achieve the separation and labeling of single cells.
  • the flux can reach 10,000 cells.
  • the droplet generating devices used in these two technologies need to be driven by electricity, and the cost is high. Each experiment can only do 4 or 8 samples at the same time, and the flexibility is limited.
  • the main purpose of this application is to provide a high-throughput single-cell chromatin accessibility sequencing method, so as to overcome the shortcomings of the prior art.
  • the embodiment of the present application provides a high-throughput single-cell chromatin accessibility sequencing method, which includes:
  • a microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet.
  • the water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase.
  • the phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;
  • the water-in-oil reaction droplets are demulsified, the DNA is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library, and then sequencing analysis is performed.
  • the sequencing method includes: lysing the cell to obtain a nucleus, and then incubating the nucleus with a transposase, so that the chromatin open zone of each nucleus is provided with a first linker sequence, and the first linker sequence It can specifically bind to the second linker sequence in the tagged primer, so that the tagged primer can capture the DNA with the first linker sequence.
  • the sequencing method specifically includes:
  • the cell nucleus and cell label after transposition are respectively made into a cell nucleus suspension and a cell label suspension;
  • a microfluidic chip which includes a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single cell sample collection port;
  • the oil as the cell isolation medium is injected into the microfluidic chip, and the cell isolation medium is brought into contact with the nucleus carrier fluid when flowing in the cell isolation medium microchannel, and the nucleus carrier fluid is sheared and wrapped to form a single cell nucleus and Single cell labelled water-in-oil reaction droplets;
  • the sequencing method includes: at least determining the position of the open chromatin region and the position of the nucleosome through sequencing analysis.
  • the embodiments of the present application also provide a method for constructing a sequencing library with high-throughput single-cell chromatin accessibility, which includes:
  • a microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet.
  • the water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase.
  • the phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;
  • Demulsification treatment is performed on the water-in-oil reaction droplets, and the DNA therein is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library.
  • the embodiment of the present application also provides a kit for constructing the sequencing library, which includes:
  • the microfluidic chip is used at least to capture and package single cell nuclei to generate water-in-oil reaction droplets.
  • the water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase, and the oil
  • the water reaction droplet contains single cell nucleus and single cell label;
  • the cell label includes a deformable microbead and a labeled primer attached to the deformable microbead, and the labeled primer can be separated from the deformable microbead under physical and/or chemical action;
  • the microfluidic chip includes a cell microfluidic channel, a cell isolation medium microfluidic channel, a cell label microfluidic channel, and a single-cell nuclear sample collection port
  • the cell microfluidic channel has a cell nuclear suspension inlet and a single cell nucleus sample collection port.
  • the cell nucleus suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single-cell nucleus suspension outlet and the cell label suspension outlet meet to make the cell microchannel
  • the output single cell nuclear suspension can be mixed with the cell label suspension output from the cell label microchannel to form a nucleus carrier liquid.
  • the flow path of the cell nucleus carrier liquid crosses the cell isolation medium microchannel, so that The cell isolation medium flowing in the microchannel of the cell isolation medium can shear and wrap the nucleus carrier fluid, thereby forming a water-in-oil reaction droplet containing a single cell nucleus and a single cell label, and the water-in-oil reaction droplet is composed of a single-cell nucleus sample Collect port output.
  • deformable microbeads such as hydrogel microbeads are used to construct cell labels, and terminal repair and supplementation are performed in water-in-oil reaction droplets.
  • an air pump is used as a power source to drive the formation of water-in-oil reaction droplets.
  • Multiple samples can be taken at the same time, which effectively improves the capture efficiency of cells, reduces the mutual contamination of microbeads after the droplets are demulsified, increases the proportion of effective data, and reduces the cost, the use is more flexible, and the operation is easier Compared with the existing indrop and dropseq platforms, the overall operation time is greatly reduced.
  • the high-throughput single-cell chromatin accessibility sequencing method provided by this application has high cell capture efficiency, high cell throughput, and high flexibility (1-8 samples can be made at the same time), Low cross-contamination, low double-packing rate, high sensitivity (detection of the chromatin open area of a single cell), low cost and other advantages, and can be fully automated, can detect small samples without power driving, and can be carried in a portable way.
  • the application prospect is broad.
  • FIG. 1 is a process flow diagram of a high-throughput single-cell chromatin accessibility sequencing process in a typical embodiment of the present application
  • FIG. 2 is a schematic structural diagram of a microfluidic chip in an exemplary embodiment of the present application
  • FIG. 3 is a schematic diagram of the generation of water-in-oil reaction droplets in a microfluidic chip in a typical embodiment of the present application
  • Fig. 4 is an optical photograph of a water-in-oil reaction droplet generated in a specific implementation case of the present application
  • Figure 5 is a Labchip detection map of DNA extracted in a specific implementation case of this application.
  • Fig. 6 is a Labchip detection map of a sequencing library in a specific implementation case of this application.
  • Figure 7 shows the location distribution of nucleosomes in a specific implementation case of the present application
  • Figure 8 shows the signal intensity of the chromatin open area in a specific implementation case of the present application
  • Figure 9 shows the open regions of chromatin on different chromosomes in a specific implementation case of the present application.
  • One aspect of the embodiments of the present application provides a high-throughput single-cell chromatin accessibility sequencing method.
  • the sequencing method includes the steps of lysing the cells, transposing the nuclei, and packaging the nuclei in a water-in-oil microreaction system (water-in-oil reaction droplets), and extracting the The steps of amplifying DNA and adding sequencing adapters to form a sequencing library, and performing sequencing analysis steps.
  • sequencing method may include:
  • a microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet.
  • the water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase.
  • the phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;
  • the water-in-oil reaction droplets are demulsified, the DNA is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library, and then sequencing analysis is performed.
  • the sequencing method includes: lysing the cell to obtain a nucleus, and then incubating the nucleus with a transposase, so that the chromatin open zone of each nucleus has a first linker sequence, and the first linker The sequence can specifically bind to the second linker sequence in the tagged primer, so that the tagged primer can capture the DNA with the first linker sequence.
  • the transposase includes Tn5 transposase.
  • the sequencing method further includes: performing physical and/or chemical treatment on the water-in-oil reaction droplet, and then incubating at 72°C-55°C to make the labeled primer capture the band DNA with the first linker sequence.
  • the collected water-in-oil reaction droplets can be irradiated with ultraviolet light to free the labeled primer (with the second linker sequence) on the microbeads and bind to the DNA with the first linker sequence.
  • the collected water-in-oil reaction droplets can also be incubated to connect the DNA therein and repair the gap.
  • the first linker sequence and the second linker sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • a connection method is adopted, so that the tagged primer sequence on the microbead can be combined with and connected to the DNA with a specific linker sequence after transposition.
  • the system used is simple, the incubation temperature is low, and the water-in-oil The stability will be higher.
  • the prior art for example, sequencing technology based on the 10X platform
  • the incubation temperature required is high, and the stability of the droplet is required. High and difficult to operate.
  • the demulsification treatment in this application preferably adopts physical demulsification methods such as ultrasound to avoid the influence of chemical components such as PFO existing in the chemical demulsification method on subsequent reactions.
  • the sequencing method specifically includes:
  • the cell nucleus and cell label after transposition are respectively made into a cell nucleus suspension and a cell label suspension;
  • a microfluidic chip which includes a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single cell sample collection port;
  • the oil as the cell isolation medium is injected into the microfluidic chip, and the cell isolation medium is brought into contact with the nucleus carrier fluid when flowing in the cell isolation medium microchannel, and the nucleus carrier fluid is sheared and wrapped to form a single cell nucleus and Single cell labelled water-in-oil reaction droplets;
  • the cell microchannel has a cell nuclear suspension inlet and a single cell nuclear suspension outlet
  • the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet
  • the single cell nuclear suspension outlet Intersect with the cell label suspension outlet, so that the single cell nucleus suspension output from the cell microchannel can be mixed with the cell label suspension output from the cell label microchannel to form a nucleus carrier liquid
  • the flow path of the cell nucleus carrier liquid Intersect the microchannels of the cell isolation medium, so that the cell isolation medium flowing through the microchannels of the cell isolation medium can shear the continuous nuclear carrier fluid into discrete droplet-shaped nuclear liquid phases and make each cell nuclear fluid
  • the phase includes a single cell nucleus and a single cell label, while the cell isolation medium is allowed to wrap the cell liquid phase to form water-in-oil reaction droplets, which are output from the single-cell sample collection port.
  • nucleus carrier liquid microfluidics (Shown as mark 13 in Fig. 2), the nucleus-carrying liquid microchannel and the cell isolation medium microchannel intersect.
  • water-in-oil reaction droplets are used as water-in-oil microreactors, and their size can be upgraded.
  • the microfluidic chip also includes a cell suspension sample cup, a cell separation medium sample cup, and a cell label that are respectively connected to the cell microchannel, cell isolation medium microchannel, and cell label microchannel. Refill cups, etc.
  • a negative pressure power generation device is provided at the single-cell nuclear sample collection port.
  • the negative pressure power generation device may use an air pump or the like, which can generate negative pressure in the microfluidic chip to drive fluid flow in each microfluidic channel.
  • an air pump can be used to extract air from the single-cell nuclear sample collection port, and a negative pressure of -4K to -10K Pa can be applied to the entire microfluidic chip.
  • a microfluidic chip in the foregoing embodiment can be referred to as shown in Figure 2, including a cell suspension sample cup 1, a cell label sample cup 2, a cell isolation medium sample cup 4, and the cell suspension
  • the liquid sample addition cup 1, the cell label sample cup 2, the cell isolation medium sample cup 4 are respectively connected with the cell microchannel 11, the cell label microchannel 12, and the cell isolation medium microchannel 14, and the microfluidic control
  • the chip is also provided with a single-cell nuclear sample collection port 3. Wherein, after the cell microchannel 11 and the cell label microchannel 12 intersect each other, they also cross the cell isolation medium microchannel 14 and further communicate with the single-cell nuclear sample collection port 3.
  • the cell microfluidic channel of the microfluidic chip is a flow channel for forming a nuclear suspension of one component of the cell nucleus liquid phase
  • the cell labeling channel is another flow channel for forming the nuclear liquid phase.
  • the flow channel of the cell label suspension of the components, the cell isolation medium micro flow channel is the flow channel of the oil phase components. All the components flow at a certain speed along with the flow channel when the pressure on the chip is increased.
  • the nucleus carrier liquid formed by mixing the cell nucleus suspension and the cell label suspension is cut by the cell isolation medium as the oil phase to form a physical isolation. By controlling the pressure and flow resistance design, the cell isolation medium can cut a single cell nucleus and cell label.
  • each water-in-oil reaction droplet serves as a micro-reaction system and contains a cell and a cell label. More intuitively, the process of forming water-in-oil reaction droplets can also refer to Figure 3, where a, b, c, and d respectively show the cell label suspension, cell nuclear suspension, cell nuclear liquid phase, and cell isolation medium. The flow direction, e shows the water-in-oil reaction droplets.
  • the cell label includes a labeled primer and a deformable bead
  • the labeled primer is attached to the deformable bead
  • the labeled primer can be physically and/or chemically Detach from deformable microbeads under the action.
  • the cell tag uses the labeled primer to identify the cell.
  • the physical and chemical effects include various physical and chemical effects well known to those skilled in the art.
  • ultraviolet light irradiation or specific enzyme cleavage can be preferably used, and it is not limited thereto.
  • the labeled primer may be labeled as an oligonucleotide chain that is ultraviolet-sensitive, light-sensitive, or can be specifically cleaved, and is not limited thereto.
  • the free labeled primer captures the target product more efficiently, and can eliminate the problem of low capture efficiency caused by the steric hindrance effect of the microbead when the primer fixed on the microbead captures the target product.
  • the tag includes a base sequence of 5-24 nt as a barcode.
  • the barcode may include 3 but not limited to 3 constant base sequences.
  • the total length of the tagged primers may range from 50 nt to 200 nt, and is not limited thereto.
  • the labeled primer may be chemically and/or physically attached to the deformable microbead.
  • the primer and the microbead can be connected by covalent bonding, chemical polymerization, antigen-antibody binding, enzyme-catalyzed connection reaction, etc., and it is not limited thereto.
  • the deformable beads can be made of organic materials or inorganic-organic composite materials, for example, polyacrylamide gel beads, agarose gel beads, agarose-coated magnetic beads, Silicon beads, microbeads made of inert materials, etc. are not limited thereto.
  • the deformable microbeads can be gel microbeads, which are beneficial to further improve the efficiency of the microfluidic chip for water-in-oil reaction droplet packaging, and cooperate with the microfluidic chip to greatly increase cell flux .
  • the mechanism may be: due to the use of deformable microbeads, each water-in-oil reaction droplet can be coated with cell-labeled microbeads, and the single packaging rate can reach 100%.
  • porous polyacrylamide microbeads are preferably used in this application, because their specific surface area is much larger than that of other microbeads, such as hard microbeads such as resin or magnetic microbeads, so the number of primers carried is far more than that of hard microbeads.
  • Other microbeads When synthesizing the polyacrylamide microbeads, the concentration of acrylamide monomer used can be 1%-10%.
  • the diameter of the microbeads may be 10 ⁇ M-200 ⁇ M.
  • the single-port double-packing rate is 1/2 of the double-port under the same cell nucleus concentration, and cell nuclei of different sizes can be adjusted by pressure (for example, with negative
  • the pressure generated by the pressure force generating device is used as the power) to realize the wrapping, especially when the negative pressure power generating device is used as the power source, the nucleus wrapping can be completed quickly and efficiently, and the gel beads are used to form the cell label.
  • the suspension flow rate has an impact force, the flow rate is controllable, and the wrapping rate can be over 90%.
  • the sequencing method further includes: constructing an amplification reaction system based on the extracted DNA and a sequencing adapter as a primer (for example, the sequence may be shown in SEQ ID NO: 3, but is not limited to this). After PCR amplification, purification is performed to obtain a sequencing library.
  • the sequencing method further includes: at least determining the position of the open chromatin region and the position of the nucleosome through sequencing analysis.
  • the sequencing method may further include a step of pre-processing the "sample to be tested" or the "sample to be tested".
  • a step of pre-processing the "sample to be tested" or the "sample to be tested".
  • applying the methods provided in the embodiments of the present application requires relatively low pre-processing. For example, preliminary enrichment can be performed according to the physical or biological characteristics of the cells, and the obtained samples can be used in subsequent steps.
  • sample to be tested or “sample to be tested” can come from an individual (such as human blood, biological tissue, etc.), or from other sources, such as some processed or unprocessed laboratory materials .
  • detection of “sample” or “sample” does not only involve diagnostic purposes, but may also involve other non-diagnostic purposes.
  • kits which is used to construct the sequencing library, and includes:
  • the microfluidic chip is used at least to capture and package single cell nuclei to generate water-in-oil reaction droplets.
  • the water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase.
  • the water reaction droplet contains single cell nucleus and single cell label;
  • the cell label includes a deformable microbead and a labeled primer attached to the deformable microbead, and the labeled primer can be separated from the deformable microbead under physical and/or chemical action;
  • composition of the cell label can be as described above, and will not be repeated here.
  • the kit further includes: at least the reagents required to purify any one or more of the extracted DNA and the sequencing library, such as magnetic beads, etc., and are not limited thereto.
  • the cell lysis reagent used can be of a type well known to those skilled in the art, and it can include any protease and protein denaturing reagents and lysis buffers that are well known to those skilled in the art that are suitable for cell lysis. System and so on.
  • the reagents required for amplifying the extracted DNA may include a sequencing adapter as a primer, a DNA polymerase and an amplification buffer well-known to those skilled in the art.
  • the main components of the amplification buffer may include: KCl, NH 4 Cl, NaCl, Tris, MgCl 2 , betaine, DMSO, water, and the like.
  • the DNA polymerase can be selected from Taq DNA polymerase, hot-start Taq polymerase, high-fidelity enzymes and the like.
  • the DNA polymerase can be any thermostable DNA polymerase known to those skilled in the art, such as: LA-Taq, rTaq, Phusion, Deep Vent, Deep Vent (exo-) , Gold 360, Platinum Taq, KAPA 2G Robust, etc., but not limited to this.
  • the transposition reagent may include a transposase, a transposase reaction buffer, a transposition reaction stop solution, etc., which are well known to those skilled in the art.
  • the transposase can be selected from Tn5 and the like, and is not limited thereto.
  • the sequencing analysis can also be performed in a manner well known to those skilled in the art, which can include basic analysis, standard analysis, and advanced analysis.
  • deformable microbeads such as hydrogel microbeads are used to construct cell labels, and terminal repair and supplementation are performed in water-in-oil reaction droplets.
  • an air pump is used as a power source to drive the formation of water-in-oil reaction droplets. It has the characteristics of high cell capture efficiency, high cell throughput, and high flexibility. It can do 1-8 samples at the same time, and can effectively reduce the mutual contamination of microbeads after droplet demulsification, low cross-contamination, and low double-packing rate.
  • the reagents and consumables used in the following examples for example: a microfluidic chip with the functions of single-cell nucleus separation and water-in-oil reaction droplet generation; the oil required to generate the reaction droplets; carrying a label Primer hydrogel beads; nuclease-free water; Taq polymerase reaction solution; PCR amplification reaction buffer; DNA amplification enzyme; transposase reaction buffer; transposase; transposition reaction termination solution; for Magnetic beads for double-stranded DNA purification; sequencing adapters for library amplification, etc., are all commercially available.
  • the purified magnetic beads used in the following embodiments may preferably be Ampure XP beads of Beckman Company, SPRI beads of Beckman Company, etc., and are not limited thereto.
  • the sequencing library construction and sequencing process of the following embodiment may include the following steps:
  • Preparation of cell lysate, reagent storage requirements The prepared cell lysate must be stored in a refrigerator at 4°C and sealed, with a shelf life of 30 days.
  • the first linker sequence (defined as linker A) can be placed in the open zone of chromatin.
  • a kind of DNA obtained can be as shown below, wherein the part marked with a single underline is the linker A, and the part marked with a double underline is the sequence on the transposase.
  • droplets On the machine to generate water-in-oil reaction droplets (hereinafter referred to as droplets)
  • the part marked with a single underline is the second linker sequence (defined as linker B), the part marked with a double underline is the sequence of a conventional sequencing primer, and the part containing J and N is a tag sequence, which may be a random sequence.
  • the collection port was irradiated with ultraviolet for 10 minutes to release the labeled primers on the beads and capture the transposed DNA.
  • step temperature time Fill in the gap 72°C 5min Annealing trap 60°C—50°C (set cooling rate 0.3°C/s) 30min
  • ATAC-I7 is shown in SEQ ID NO: 3.
  • the sequencing process adopts the PE150 sequencing solution, which can be performed on platforms such as Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, and Illumina Miseq.
  • platforms such as Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, and Illumina Miseq.
  • the corresponding operation methods and experimental conditions are well known to those skilled in the art.
  • the corresponding sequencing analysis can be seen in Figure 7-9.
  • paired-end ATAC-Seq sequencing reads can reflect the information of nucleosome packaging and positioning.
  • the insert length of the sequencing reads will show a periodic distribution of approximately 200bp.
  • Figure 8 for the ATAC-Seq signal intensity of the 3kb region upstream and downstream of the transcription start site (TSS).
  • Figure 9 shows the number and distribution of peaks on the chromosome.
  • the inventor of this application also uses the same cell nucleus sample as in this example, based on some existing sequencing solutions, such as: a typical high-quality traditional ontology ATAC-seq solution; Cusanovich et al., Science, May 22, 2015 ; 348(6237):910-14; Buenrostro et al., Nature, July 23, 2015; 523(7561):486-90; The ideal sequencing metric for ATAC-seq experiments, a controlled experiment was carried out.
  • some existing sequencing solutions such as: a typical high-quality traditional ontology ATAC-seq solution; Cusanovich et al., Science, May 22, 2015 ; 348(6237):910-14; Buenrostro et al., Nature, July 23, 2015; 523(7561):486-90; The ideal sequencing metric for ATAC-seq experiments, a controlled experiment was carried out.

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Abstract

L'invention concerne un procédé de séquençage haut débit pour l'accessibilité de chromatines monocellulaires, comprenant les étapes suivantes : incubation d'un noyau cellulaire à l'aide d'une transposase, puis l'emballage du noyau cellulaire soumis à une transposition et un marqueur cellulaire dans une gouttelette de réaction eau dans huile à l'aide d'une puce microfluidique ; soumission de la gouttelette de réaction eau-dans-huile à un traitement tel qu'une irradiation UV, de telle sorte qu'une amorce avec un marqueur à l'intérieur de cette dernière est libéré à partir de billes déformables ; et incubation et désémulsification de la gouttelette de réaction eau-dans-huile, puis la génération d'une banque de séquençage, et enfin réalisation d'une analyse de séquençage. Ledit procédé de séquençage présente les avantages d'une efficacité de capture de cellules élevée, un haut débit cellulaire, une grande flexibilité, une faible contamination croisée, un faible taux d'encapsidation double, une sensibilité élevée et un faible coût ; il peut être entièrement automatisé, n'a pas besoin d'être entraîné par l'électricité, et est portable, fournissant une large perspective d'applications.
PCT/CN2019/128154 2019-12-25 2019-12-25 Procédé de séquençage haut débit pour l'accessibilité de chromatines monocellulaires Ceased WO2021128034A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024186877A1 (fr) * 2023-03-07 2024-09-12 Board Of Regents, The University Of Texas System Procédés et compositions pour l'amplification et le séquençage du génome et de l'épigénome

Citations (3)

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