A microwell chip and single cell analysis method

Abstract

The present invention discloses a microporous chip and a single-cell analysis method. The microporous chip is provided with a plurality of double-pass penetrating micropores, and the micropores are used to load a single-cell suspension or a single-cell nuclear suspension of a sample to be tested and microbeads with nucleic acid molecules of known base sequences. The single-cell analysis method comprises the following steps: S1, mixing a single-cell suspension or a single-cell nuclear suspension of a tissue sample to be tested with microbeads with nucleic acid molecules of known base sequences and loading the mixture into the micropores of the microporous chip; S2, polymerizing and amplifying nucleic acid molecules on the microporous chip in S1 to construct a single-cell sequencing library. The microporous chip and the single-cell analysis method of the present invention can be applied to high-throughput single-cell multi-omics sequencing, and can obtain specific omics information such as full-length transcriptomes, epigenomes, and proteomes of hundreds of thousands to millions of single cells in batches at one time.

Description

Technical Field

[0001] The present application relates to the field of gene sequencing technology, and in particular to a microporous chip and a single-cell analysis method. Background Art

[0002] Single-cell sequencing mainly relies on microfluidics or micropore systems to lock individual cells into separate reaction spaces, and then construct a second-generation sequencing library to achieve the goal of obtaining thousands to tens of thousands of cells in one experiment. Taking the 10× microfluidic platform as an example, when the amount of cell input exceeds the recommended loading amount, there will be two or more cells in a droplet at the same time, resulting in multiple cells in the same droplet being unable to be distinguished in subsequent analysis. Based on this, in order to control only one cell in each droplet, the loading concentration of the cell must be strictly controlled, but this will cause most of the droplets to have no cells, forming an "empty load", greatly reducing the experimental throughput and cell utilization. At the same time, the cells on the microfluidics-based sequencing platform are not tested in parallel, and the batch effect problem is more significant. In addition, there are disadvantages such as expensive equipment, difficulty in carrying, and high sequencing costs.

[0003] In the prior art, the principle of microwell single-cell sequencing methods is generally to use gravity sedimentation to capture single cells and single microbeads into microwells. The Poisson distribution characteristics of microwell gravity sedimentation limit the efficiency of cell detection. The double-pass microwell array chip used in some digital PCR systems is based on the liquid absorption characteristics of capillary action, which can circumvent the inefficiency problems caused by gravity sedimentation and Poisson distribution. However, the thickness and material of the double-pass microwell array chip used in the digital PCR system are not suitable for direct application to single-cell omics methods, and the uniform liquid capillary action will cause multiple microbeads or multiple cells to be contained in the microwell, making it impossible to directly analyze single-cell resolution omics data. Summary of the invention

[0004] Based on this, it is necessary to provide a microporous chip and a single-cell analysis method. The single-cell analysis method of the present invention can be applied to high-throughput single-cell multi-omics sequencing, and can obtain full-length transcriptome, epigenome, proteome and other specific omics information of hundreds of thousands to millions of single cells in batches at one time. The single-cell analysis method of the present invention can be applied to fresh, frozen or embedded tissues, and is not limited by cell activity and state.

[0005] An embodiment of the present application provides a microporous chip.

[0006] A microporous chip is provided with a plurality of double-pass penetrating micropores, wherein the micropores are used to load single cell suspension or single cell nucleus suspension of a sample to be tested and microbeads with nucleic acid molecules with known base sequences.

[0007] In some embodiments, the microporous chip further satisfies at least one of the following conditions:

[0008] (1) The shape of the micropores includes one or more of circular, elliptical, and polygonal;

[0009] (2) The pore size of the micropore is 10 μm to 1 mm, preferably, the pore size of the micropore is 20 μm to 100 μm;

[0010] (3) The microporous chip is generally circular, trapezoidal or rectangular;

[0011] (4) The material of the microporous chip includes any one of glass fiber, polymer, and silicon wafer;

[0012] (5) The microbeads loaded in the microporous chip include any one of polymer microbeads, magnetic microbeads, hydrogel microbeads and degradable polymer microbeads;

[0013] (6) The outer surface of the microporous chip is treated to be hydrophobic, and the inner wall of the microporous is treated to be hydrophilic.

[0014] An embodiment of the present application also provides a single cell analysis method.

[0015] A single cell analysis method, using the microporous chip, comprises the following steps:

[0016] S1. Mixing a single cell suspension or a single cell nucleus suspension of a tissue sample to be tested with microbeads with nucleic acid molecules of known base sequences and loading the mixture into microwells of a microwell chip;

[0017] S2. Performing nucleic acid molecule polymerization and amplification on the microporous chip in S1 to construct a single-cell sequencing library.

[0018] In some of the embodiments, step S1 of the single cell analysis method further includes the following step: using a fixative to fix the single cells or cell nuclei in the single cell suspension or single cell nucleus suspension.

[0019] In some embodiments, the fixing liquid includes one or more of an aldehyde fixing liquid, an alcohol fixing liquid, and an acid fixing liquid.

[0020] In some of the embodiments, in step S1 of the single cell analysis method, different microwells on the microwell chip are loaded with different samples to be tested.

[0021] In some of the embodiments, in step S1 of the single cell analysis method, when the nucleic acid molecule of known base sequence on the microbead is hybridized with the single cell or single cell nucleus, the in situ labeled object in the single cell or single cell nucleus is RNA or DNA, and correspondingly, the nucleic acid molecule of known base sequence on the microbead includes nucleic acid;

[0022] Alternatively, the in situ labeling object in a single cell or a single cell nucleus is a protein, and correspondingly, the nucleic acid molecules with known base sequences on the microbeads include nucleic acids modified on antibodies.

[0023] In some of the embodiments, when the in situ labeling object in a single cell or a single cell nucleus is RNA or protein, the nucleic acid molecule with a known base sequence on the microbead is a single-stranded oligonucleotide with a known base sequence; when the in situ labeling object in a single cell or a single cell nucleus is DNA, the nucleic acid molecule with a known base sequence on the microbead is a transposase linker sequence with a known base sequence.

[0024] In some embodiments, in step S1 of the single cell analysis method, the nucleic acid molecule with a known base sequence on the microbead includes a conditional cleavable site, wherein the conditional cleavable site includes any one of: dU base modification, disulfide bond modification, photocleavable linker, and restriction endonuclease recognition sequence;

[0025] And / or, the microbeads are directly coupled to the nucleic acid molecules of known base sequences by any one of: condensation acylation reaction based on carboxyl amino groups, cross-linking reaction based on biotin and streptavidin, ester bond connection, disulfide bond connection, and dipole interaction.

[0026] In some of the embodiments, in step S1 of the single-cell analysis method, when the single-cell suspension or single-cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence are loaded into the microwells of the microporous chip, the method includes the following steps: the single-cell suspension or single-cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence are adsorbed into the microwells along with the liquid on the surface of the microporous chip through capillary action, wherein each of the microwells can accommodate multiple microbeads.

[0027] In some of the embodiments, in step S1, a single cell suspension or a single cell nucleus suspension of the tissue sample to be tested is obtained by the following steps: subjecting the cell line of the sample to be tested to a lysis reaction with a lysis solution, terminating the lysis after a predetermined lysis time, and obtaining a single cell nucleus of the sample to be tested.

[0028] In some of the embodiments, in step S2 of the single cell analysis method, when the nucleic acid molecules are polymerized and amplified on the microporous chip in S1, the amplification method includes any one of a multi-step PCR amplification method and a multi-step ligase ligation method.

[0029] In some of the embodiments, in step S2 of the single-cell analysis method, the amplification method includes split-pool segmented PCR hybridization extension amplification and split-pool segmented ligase ligation extension.

[0030] In some of the embodiments, in step S2 of the single-cell analysis method, after the single-cell suspension or single-cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules with known base sequences are loaded onto the chip, the evaporation of the liquid on the surface of the microporous chip is promoted by natural evaporation or vacuum evaporation.

[0031] In some of the embodiments, in step S2 of the single-cell analysis method, when the nucleic acid molecules are polymerized and amplified on the microporous chip in S1, the following steps are included: the microporous chip is sealed with sealing oil, and the in situ labeled objects in the single cell or single cell nucleus are released by constant temperature heating to hybridize with the nucleic acid molecules of known base sequence on the microbeads.

[0032] In some of the embodiments, in step S2 of the single cell analysis method, the amplification method includes any one of exponential PCR amplification and linear PCR amplification.

[0033] In some of these embodiments, the single-cell sequencing library of the single-cell analysis method includes single-cell transcriptome data and / or chromatin accessibility genomic data.

[0034] In some of the embodiments, the single-cell analysis method further comprises the following step: S3, sequencing the single-cell sequencing library.

[0035] The above-mentioned microporous chip can be applied to high-throughput single-cell multi-omics sequencing, and can provide high-throughput parallel single-cell sequencing, with simple equipment requirements, simple operation procedures, and easy portability.

[0036] In the above-mentioned single-cell analysis method, by introducing the first section of cell identity label on the in-situ labeling object of a single cell or a single cell nucleus, at least one microbead and at least one single cell or a single cell nucleus are loaded into the microporous chip, and they are uniformly and quickly sucked into the micropores of the microporous chip through capillary action, and the second section of cell identity label on the microbead is combined with the nucleic acid of the single cell or a single cell nucleus to perform nucleic acid polymerization and amplification reaction. After the microbeads and the single cell or a single cell nucleus are loaded into the microporous chip, the evaporation of the liquid on the surface of the microporous chip can be promoted by natural evaporation, vacuum evaporation, etc., so as to add new reaction reagents to realize convenient multiple rounds of reactions in the micropores, while making the reaction compartments between the micropores more independent and reducing molecular contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0038] In order to more completely understand the present application and its beneficial effects, the following description will be given in conjunction with the accompanying drawings. In the following description, the same reference numerals represent the same parts.

[0039] Figure 1 A schematic diagram of the experimental process in one embodiment of the present invention;

[0040] Figure 2 This is a schematic diagram of the process of using a single cell nucleus suspension to perform a transcriptome experiment in one embodiment of the present invention;

[0041] Figure 3 This is a schematic diagram of the process of performing epigenomic experiments using a single cell nucleus suspension in one embodiment of the present invention;

[0042] Figure 4 Schematic diagram of the distribution of microbeads and cell nuclei captured in micropores of a chip through capillary action in one embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0044] In the description of the present invention, “several” means more than one, “multiple” means more than two, “greater than”, “less than”, “exceed”, etc. are understood as excluding the number, and “above”, “below”, “within”, etc. are understood as including the number.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0046] The present application embodiment provides a microporous chip and a single-cell analysis method to solve the problems of low throughput and low cell utilization in single-cell sequencing experiments in traditional technologies; and at least one of the problems of significant batch effect, difficulty in carrying, and high sequencing costs. The single-cell analysis method of the present application can be used for high-throughput single-cell multi-omics sequencing, such as for single-cell transcriptome sequencing and chromatin accessibility sequencing. It is suitable for fresh, frozen or embedded tissues during sequencing, is not limited by cell activity and state, and can obtain full-length transcriptomes, epigenomes, proteomes and other specific omics information of hundreds of thousands or even millions of single cells in batches at one time.

[0047] A microporous chip is provided with a plurality of double-pass penetrating micropores, wherein the micropores are used to load a single cell suspension or a single cell nucleus suspension of a sample to be tested and microbeads with nucleic acid molecules of known base sequences.

[0048] In some embodiments, the shape of the micropores includes one or more of circular, elliptical, and polygonal shapes.

[0049] In some embodiments, the pore size of the micropore is 10 μm to 1 mm. Preferably, the pore size of the micropore is 20 μm to 100 μm. More preferably, the pore size of the micropore is 40 μm to 60 μm. Specifically, different micropore shapes or sizes can be selected according to the needs of single cell or single cell nucleus, molecular size, and micropore spacing process adjustment or modification on the surface of the micropore chip.

[0050] In some embodiments, the microporous chip is circular, trapezoidal or rectangular in shape as a whole. When in use, the corresponding microporous chip can be used to perform large-scale ultra-high throughput single cell or single cell nucleus analysis.

[0051] In some embodiments, the material of the microporous chip includes any one of glass fiber, high molecular polymer, and silicon wafer. Specifically, different materials can be selected for preparing the microporous chip according to the requirements of the double-pass etching process corresponding to the size and thickness of the microporous chip.

[0052] In some embodiments, the microbeads loaded in the microporous chip include any one of polymer microbeads, magnetic microbeads, hydrogel microbeads and degradable polymer microbeads. Specifically, nucleic acid molecules can be cross-linked to the surface of microbeads or polymerized inside microbeads according to needs, and the diameter of microbeads can be adaptively changed according to the size of micropores.

[0053] In some embodiments, the outer surface of the microporous chip is treated hydrophobically, and the inner wall of the microporous is treated hydrophilically.

[0054] The above-mentioned microporous chip can be applied to high-throughput single-cell multi-omics sequencing, and can provide high-throughput parallel single-cell sequencing, with simple equipment requirements, simple operation procedures, and easy portability.

[0055] An embodiment of the present application also provides a single cell analysis method.

[0056] A single cell analysis method, using the microporous chip, comprises the following steps:

[0057] S1. Mixing a single cell suspension or a single cell nucleus suspension of a tissue sample to be tested with microbeads with nucleic acid molecules of known base sequences and loading the mixture into microwells of a microwell chip;

[0058] S2. Performing nucleic acid molecule polymerization and amplification on the microporous chip in S1 to construct a single-cell sequencing library.

[0059] In some embodiments, in step S1, when the single cell suspension or single cell nucleus suspension of the tissue sample to be tested is mixed with microbeads with nucleic acid molecules of known base sequences, the following steps are specifically included: adding different nucleic acid molecules or proteins of known base sequences to different wells of the multi-well plate, and the nucleic acid molecules or proteins with the same known base sequence in the same well can achieve in situ labeling of the object inside the single cell or single cell nucleus for RNA reverse transcription or epigenome disruption, while connecting the single cell or single cell nucleus with the nucleic acid molecule of known base sequence as the first segment of cell identity label, so as to facilitate the rapid and accurate determination of single cells or single cell nuclei from different samples in the subsequent analysis process. Among them, the multi-well plate can be a 96-well plate.

[0060] In some embodiments, step S1 of the single cell analysis method further includes the following step: fixing the single cell or cell nucleus in the single cell suspension or single cell nucleus suspension using a fixative. It should be noted that in chromatin accessibility sequencing, the cell nucleus may not be fixed.

[0061] In some embodiments, the fixing liquid includes one or more of an aldehyde fixing liquid, an alcohol fixing liquid, and an acid fixing liquid.

[0062] In some embodiments, the aldehyde fixative includes, for example, paraformaldehyde.

[0063] In some embodiments, the alcoholic fixing solution includes ethanol.

[0064] In some of the embodiments, in step S1 of the single cell analysis method, different microwells on the microwell chip are loaded with different samples to be tested.

[0065] In some embodiments, in step S1 of the single cell analysis method, when the nucleic acid molecules with known base sequences on the microbeads are hybridized with single cells or single cell nuclei, the in situ labeled object in the single cells or single cell nuclei is RNA or DNA, and correspondingly, the nucleic acid molecules with known base sequences on the microbeads include nucleic acids. For example, the nucleic acid molecules with known base sequences on the microbead surface carrying oligo-dT can be hybridized with RNA in the treated single cells or single cell nuclei; the fixed hybridization sequences carried by the nucleic acid molecules with known base sequences on the microbead surface can be hybridized with DNA in the treated single cells or single cell nuclei.

[0066] In some of the embodiments, the in situ labeling target in a single cell or a single cell nucleus is a protein, and correspondingly, the nucleic acid molecule with a known base sequence on the microbead includes a nucleic acid modified on an antibody.

[0067] In some embodiments, when the in situ labeling object in a single cell or a single cell nucleus is RNA or protein, the nucleic acid molecule of known base sequence on the microbead is a single-stranded oligonucleotide of known base sequence. When the in situ labeling object in a single cell or a single cell nucleus is DNA, the nucleic acid molecule of known base sequence on the microbead is a transposase adapter sequence of known base sequence.

[0068] In some of the embodiments, in step S1 of the single-cell analysis method, the nucleic acid molecule of known base sequence on the microbead includes a conditional breakable site, wherein the conditional breakable site includes: any one of dU base modification, disulfide bond modification, photocleavable linker and restriction endonuclease recognition sequence; specifically, different breakage modes can be selected as needed to facilitate the release of nucleic acid molecules into the microwells for reaction.

[0069] In some embodiments, the microbeads are directly coupled to nucleic acid molecules of known base sequences in any of the following ways: condensation acylation reaction based on carboxyl amino groups, cross-linking reaction based on biotin and streptavidin, ester bond connection, disulfide bond connection, and dipole interaction. Specifically, different coupling methods can be selected as needed to facilitate the release of nucleic acid molecules into microwells for reaction.

[0070] For example, in one embodiment, the microbead surface comprises a carboxyl modification, the nucleic acid molecule of known base sequence comprises an amino modification, and the conditional cleavable site comprises a dU base modification. The nucleic acid molecule of known base sequence is attached to the microbead surface by an acylation reaction, and the nucleic acid molecule is released into the microporous liquid environment for reaction by cleavage of the dU base.

[0071] In some embodiments, in step S1 of the single cell analysis method, when loading the single cell suspension or single cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence into the micropores of the microporous chip, the following steps are included: the single cell suspension or single cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence are adsorbed into the micropores along with the liquid on the surface of the microporous chip by capillary action, rather than gravity sedimentation and Poisson distribution. Wherein, each of the micropores can accommodate multiple microbeads.

[0072] In some of the embodiments, in step S1, a single cell suspension or a single cell nucleus suspension of the tissue sample to be tested is obtained by the following steps: subjecting the cell line of the sample to be tested to a lysis reaction with a lysis solution, terminating the lysis after a predetermined lysis time, and obtaining a single cell nucleus of the sample to be tested.

[0073] In some of the embodiments, in step S2 of the single cell analysis method, when the nucleic acid molecules are polymerized and amplified on the microporous chip in S1, the amplification method includes any one of a multi-step PCR amplification method and a multi-step ligase ligation method.

[0074] In some of the embodiments, in step S2 of the single-cell analysis method, the amplification method includes split-pool segmented PCR hybridization extension amplification and split-pool segmented ligase ligation extension.

[0075] In some embodiments, in step S2 of the single cell analysis method, after the single cell suspension or single cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules with known base sequences are loaded onto the chip, the evaporation of the liquid on the surface of the microporous chip is promoted by natural evaporation or vacuum evaporation, thereby adding new reaction reagents to realize convenient multiple rounds of reactions in the microwells, while making the reaction compartments between the microwells more independent and reducing molecular contamination.

[0076] In some of the embodiments, in step S2 of the single-cell analysis method, when the nucleic acid molecules are polymerized and amplified on the microporous chip in S1, the following steps are included: the microporous chip is sealed with sealing oil, and the in situ labeled objects in the single cell or single cell nucleus are released by constant temperature heating to hybridize with the nucleic acid molecules of known base sequence on the microbeads.

[0077] In some of the embodiments, in step S2 of the single cell analysis method, the amplification method includes any one of exponential PCR amplification and linear PCR amplification.

[0078] In some of these embodiments, the single-cell sequencing library of the single-cell analysis method includes single-cell transcriptome data and / or chromatin accessibility genomic data.

[0079] In some of the embodiments, the single-cell analysis method further comprises the following step: S3, sequencing the single-cell sequencing library.

[0080] In some of the embodiments, in step S1, when constructing a transcriptome sequencing library, the nucleic acid molecule with a known base sequence is an oligonucleotide sequence, which can be randomly hybridized and paired with any region of the nucleic acid, and then reverse transcription reaction of the full-length region of the nucleic acid is performed without bias; the primer sequence of the nucleic acid molecule connected to the microbeads of the transcriptome sequencing library includes four parts: a library adapter sequence, a cell tag formed by a combination of three segments of 96 fixed sequences, a molecular tag (UMI, unique molecular identifier) ​​and a Poly (dT) sequence. The library adapter sequence is used for subsequent sequencing on the machine; the cell tag sequence with oligonucleotides on the cell nucleus (the first segment of the cell identity tag) and the cell tag formed by the combination of three segments of 96 fixed sequences on the microbeads (the second segment of the cell identity tag) are combined to identify different cells; UMI is a sequence composed of random bases, and each DNA molecule contains a unique UMI to identify different DNA molecules during mixed sequencing; the Poly (dT) sequence is used to capture cDNA molecules containing polyA tails.

[0081] In some of the embodiments, in step S1, when constructing the epigenomic sequencing library, the nucleic acid molecules with known base sequences use the transposase adapter sequence containing the cell identity tag sequence to interrupt the chromatin open region; the primer sequence for the microbead connection of the epigenomic sequencing library includes four parts: the library adapter sequence, the cell tag formed by the combination of three segments of 96 fixed sequences, the UMI and the transposase adapter sequence 1. The library adapter sequence is used for subsequent sequencing on the machine; the oligonucleotide cell tag sequence on the cell nucleus (the first segment of the cell identity tag) and the cell identity tag sequence on the microbead (the second segment of the cell identity tag) are combined to identify different cells; the UMI is used to identify different DNA molecules during mixed sequencing; and the transposase adapter sequence 1 is used to capture the DNA sequence interrupted by the transposase.

[0082] In some embodiments, in step S1, when a single cell suspension or a single cell nucleus suspension of a tissue sample to be tested is mixed with microbeads with nucleic acid molecules of known base sequences, a split-pool strategy of "disperse-merge-disperse" is adopted to prepare microbeads labeled with multiple nucleic acids, specifically comprising the following steps:

[0083] Design a nucleic acid molecule with a known base sequence, divide the nucleic acid molecule with a known base sequence into three segments, and set a PCR hybridization adapter sequence between two adjacent segments, wherein the first segment starting from the 5' end includes an amino modification, a fixed library adapter sequence and a partial cell identity tag sequence. For the construction of the transcriptome sequencing library, the 3' end contains a partial cell tag sequence, a molecular tag sequence, and a poly T tail; for the construction of the epigenome sequencing library, the 3' end contains a partial cell tag sequence, a molecular tag sequence, and a transposase adapter sequence 2. Among them, a predetermined number of X sequences are designed for the cell tag sequence portion of all sequences, each of which is placed independently; an amine group is used to replace the hydroxyl group at the C6 position of the nucleotide at the 5' end of the first segment sequence. Equal amounts of carboxyl-modified microbeads are coupled to the X first segment sequences respectively, and then X modified microbeads are collected and obtained, mixed evenly, and then divided into X equal parts, mixed with the X second segment sequences, and then PCR sequence extension is performed, and then divided into X equal parts, mixed with the X third segment sequences, and then PCR sequence extension is performed, and then denatured and melted to obtain X 3 A single-stranded oligonucleotide-modified microbead, X 3 Micro beads.

[0084] Example 1

[0085] This embodiment provides a single cell analysis method using the above-mentioned microporous chip.

[0086] A single cell analysis method comprises the following steps.

[0087] (1) Sample preparation: according to Figure 1 The method flow shown in the figure uses human 293T cell line and mouse 3T3 cell line to construct single-cell transcriptome sequencing library. The main process of library structure construction is as follows Figure 2 shown.

[0088] The digested human 293T cell line and mouse 3T3 cell line were washed with 1×PBS solution, resuspended with 1 mL of RSBT solution containing 0.1% IGEPAL CA-630, placed on ice for 3 min, and 5 mL of RSBT solution was added to terminate the lysis reaction to obtain human 293T cell nuclei and mouse 3T3 cell nuclei. The RSBT solution includes 10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and 1% RNase inhibitor.

[0089] The split-pool strategy of "disperse-merge-disperse" was adopted to prepare microbeads with multiple nucleic acid tags, which specifically included the following steps: designing nucleic acid molecules with known base sequences, dividing the nucleic acid molecules with known base sequences (as the second segment cell identity tag) into three segments, and setting PCR hybridization adapter sequences between two adjacent segments, wherein the first segment starting from the 5' end includes amino modification, fixed library adapter sequence ( Figure 2 In the representation: library adapter sequence 2) and partial cell identity tag sequence ( Figure 2 In the text, it is represented as: cell tag 2). For the construction of transcriptome sequencing library, the 3' end contains part of the cell tag sequence, molecular tag sequence, and poly T tail, see Figure 2 As shown; for the construction of epigenomic sequencing library, the 3' end contains part of the cell tag sequence, molecular tag sequence, and transposase adapter sequence 2, see Figure 3 As shown. Among all the sequences, 96 sequences were designed for the cell tag sequence, each of which was placed independently; an amine group was used to replace the hydroxyl group at the C6 position of the nucleotide at the 5' end of the first sequence. Equal amounts of carboxyl-modified microbeads were coupled with 96 first-segment sequences respectively, and then 96 modified microbeads were collected and mixed evenly, and then divided into 96 equal parts, mixed with 96 second-segment sequences, and then PCR sequence extension was performed, and then divided into 96 equal parts, mixed with 96 third-segment sequences, and then PCR sequence extension was performed, and then denatured and melted to obtain microbeads with 884,736 single-stranded oligonucleotide modifications, that is, 884,736 microbeads containing nucleic acid molecules with known base sequences.

[0090] (2) Reverse transcription: The human 293T cell nuclei and mouse 3T3 cell nuclei prepared in step (1) were fixed with 4% PFA, resuspended with PBST, and then added with reverse transcription reagents containing reverse transcriptase, reverse transcription reaction buffer, dNTPs, reverse transcription random primers, RNase inhibitor, 10% TritonX-10, and 50% PEG8000, mixed evenly, and then evenly distributed into 96-well plates for reverse transcription reaction. After the reverse transcription reaction was completed, 10 μL of 40 mM EDTA was added to each of the 96 wells and incubated at 37° C. for 15 min to terminate the reverse transcription reaction, and then washed 3 times with 3×SSC and 1 time with PBS.

[0091] (3) Exocleavage: Add an exonuclease reagent containing EXO I exonuclease, exonuclease reaction buffer, and RNase inhibitor to the reaction product after the reverse transcription reaction after washing in step (2), incubate at 37°C for 30 min for exocleolysis to remove excess random primers, and then wash three times with 3×SSC and once with PBS.

[0092] (4) Connecting the capture linker: Add the exosome after washing in step (3) to a reaction system containing terminal transferase, terminal transferase reaction buffer, RNase inhibitor, and dATP, incubate at 37° C. for 30 min, and wash three times with PBST after the reaction.

[0093] (5) Amplification reaction: A hexagonal double-pass microwell chip was selected. The thickness of the microwell chip was 800 μm, the diagonal length of the microwell chip was 60 μm, and the microwell spacing on the microwell chip was 15 μm. Take 5 μL of the mixed 293T cell nuclei and mouse 3T3 cell nuclei (about 4 million / mL) prepared in step (4) and resuspend them in about 30,000 molecular labeled microbeads. Then add 1 μL of P7 adapter primer and 5 μL of 2× high-fidelity polymerase. After mixing, load them onto the microwell chip. The capture distribution of microbeads and cell nuclei is shown in the following figure. Figure 4 As shown, the cell nucleus and microbeads in the microwell chip have a hole-falling rate of more than 70%. Then, the amplification system containing constant temperature polymerase, RNase H, USER enzyme and high-fidelity polymerase is evenly added to the reverse side of the microwell chip after the first sample addition. The microwell chip is placed vertically in a centrifuge tube, and sealing oil is added to seal the microwells so that each microwell forms a separate reaction space. The microwell chip is then placed in a PCR thermal cycler for pre-amplification reaction, see Figure 1 shown.

[0094] (6) Collecting liquid and purifying: After the pre-amplification reaction in step (5) is completed, the liquid and microbeads in the microporous chip are fully collected by multiple centrifugation, and the collected liquid and microbeads are placed on a magnetic rack, and the supernatant is transferred to a new reaction tube. The total volume V1 of the supernatant is measured with a pipette, and then DNA Clean Beads purification magnetic beads are added, and the amount of purification magnetic beads added is 1.5 times V1, and the cDNA solution is purified.

[0095] (7) Sequencing library amplification: The cDNA solution obtained in step (6) was added to an amplification system containing index primers P5 and P7 adapter primers and a high-fidelity polymerase for amplification to obtain a sequencing library with an index. The library was then purified using DNAClean Beads purification magnetic beads to obtain a sequencing library. The library concentration was determined using Qubit 3.0 fluorescent reagent and stored at -20°C.

[0096] (8) Sequencing the sequencing library obtained in step (7).

[0097] Example 2

[0098] This embodiment provides a single cell analysis method using the above-mentioned microporous chip.

[0099] A single cell analysis method comprises the following steps.

[0100] (1) Sample and reagent preparation: according to Figure 1 The method flow shown in the figure uses human 293T cell line and mouse 3T3 cell line to construct single-cell chromatin accessibility epigenome sequencing library. The main process of library structure construction is as follows Figure 2 shown.

[0101] The transposase-embedded recognition fixed sequence ( Figure 3 In the expression: library adapter sequence 1) and cell identity tag sequence (as the first cell identity tag, Figure 3 The primers of cell label 1 (which includes 384 combinations) were placed in a PCR instrument at 95°C for 2 minutes and cooled to 25°C at a rate of 0.1°C / second. They were diluted with enzyme-free water and dispensed into multiple 96-well plates. A mixed reagent containing Tn5 transposase, coupling buffer and dilution buffer was added, mixed thoroughly, incubated at 30°C for 1 hour to form a primer working solution, and stored in a -20°C refrigerator after incubation for later use.

[0102] The digested human 293T cell line and mouse 3T3 cell line were washed with 1×PBS solution, respectively, and then resuspended with 1 mL of lysis solution containing 0.1% IGEPAL CA-630 RSBT washing solution, placed on ice for lysis for 3 minutes, and 5 mL of RSBT washing solution was added to terminate the lysis reaction, and human 293T cell nuclei and mouse 3T3 cell nuclei were obtained for use. Among them, RSBT washing solution includes 10mM Tris-HCl with a pH value of 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 and 1% RNase inhibitor.

[0103] (2) Transposition enzyme digestion reaction: The mixed human 293T cell nuclei and mouse 3T3 cell nuclei were evenly distributed into a 96-well plate, and then the enzyme digestion system containing the primer working solution stored at -20°C in step (1), 2× enzyme digestion reaction solution, 1% digitonin, 10% Tween-20 and 1×PBS was added to each well of the 96-well plate. After being fully mixed, the plate was placed at 55°C for half an hour. After the reaction was completed, the 96-well plate was taken out and placed on ice for 5 minutes to terminate the enzyme digestion reaction. The cells in the 96-well plate were collected with a spray gun, centrifuged at 500g / 5min, and then washed twice with RSBT washing solution. The RSBT washing solution includes 10mM Tris-HCl with a pH value of 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 and 1% RNase inhibitor.

[0104] (3) Release the enzyme-cut fragments: Select a hexagonal double-pass microporous chip with a thickness of 800 μm, a diagonal length of 60 μm, and a micropore spacing of 15 μm. Take 5 μL of the cell nuclei (about 4 million / mL) washed in step (2) and resuspend them in about 30,000 molecular-labeled magnetic beads, then add 7 μL of 50 mM EDTA and 5 μL of 2× high-fidelity polymerase, mix well and load them onto the microporous chip, so that the cell nuclei and microbeads in the microporous chip have a pore-falling rate greater than 70%, place the microporous chip vertically in a centrifuge tube, fasten the tube cover, and then place the centrifuge tube at 50°C for 30 minutes to release the genomic fragments interrupted by enzyme cutting, and place the microporous chip in a PCR thermal cycler for amplification reaction.

[0105] (4) Fragment release and amplification reaction: Take 30,000-40,000 molecular-labeled microbeads, wash them twice with 1×PBS, and place them on ice for later use. After adjusting the cell density after enzyme digestion in step (3) to 5 million / mL, take 4 μL of the enzyme-digested cells and resuspend them in the washed microbeads to form a mixed system. Take 14 μL of the prepared mixed system and blow it evenly. Use a gun tip to quickly and evenly load it onto the microporous chip. Use the gun tip to gently scrape the surface until the liquid is completely absorbed. Use a vacuum pump to gently extract the liquid on the surface of the microporous chip so that the liquid on the microporous surface of the microporous chip is concave. Place the microporous chip in a centrifuge tube, tighten the tube cap, and place it at 50°C for 30 minutes to release the genomic fragments interrupted by enzyme digestion. Take out the microwell chip from the centrifuge tube, add amplification reagent evenly to the reverse side of the microwell chip where the sample was first added, scrape the surface with a gun tip until the liquid is completely absorbed, place the microwell chip vertically in a new centrifuge tube, add sealing oil to seal the microwells so that each microwell forms a separate reaction space, and then place the microwell chip in a PCR thermal cycler for pre-amplification reaction. Figure 1 shown.

[0106] (5) Collecting liquid and purifying: After the pre-amplification reaction in step (4) is completed, the liquid and microbeads in the microporous chip are fully collected by multiple centrifugation, and the collected liquid and microbeads are placed on a magnetic rack. The supernatant is aspirated and transferred to a new reaction tube, and the total volume V2 of the supernatant is measured with a pipette. Then, DNA Clean Beads purification magnetic beads are added, and the amount of purification magnetic beads added is 1.5 times V2. Purification is performed to obtain a cDNA solution.

[0107] (6) Sequencing library amplification: The cDNA solution obtained in step (5) was added to an amplification system containing the index primers P5 and P7 adapter primers and a high-fidelity polymerase for amplification reaction to obtain a sequencing library with an index. The library was then purified using DNA Clean Beads purification magnetic beads to obtain a sequencing library. The library concentration was determined using Qubit 3.0 fluorescent reagent and stored at -20°C.

[0108] (7) Sequencing the sequencing library obtained in step (6).

[0109] In summary, in the single-cell analysis method of the present invention, by introducing the first section of cell identity tags on the in situ labeled object of a single cell or a single cell nucleus, at least one microbead and at least one single cell or a single cell nucleus are loaded into a microporous chip, and the microbeads are uniformly and quickly sucked into the micropores of the microporous chip through capillary action, and the second section of cell identity tags on the microbeads are combined with the nucleic acid of the single cell or single cell nucleus to perform nucleic acid polymerization and amplification reactions. The present invention utilizes the in situ labeling of single cells or single cell nuclei and the capillary action of the microporous chip to quickly and efficiently capture and isolate a large number of single cells, then amplify the genetic information of the single cells in one step, construct a sequencing library, and then perform sequencing, thereby realizing high-throughput detection of multi-omics genetic information at the single cell level.

[0110] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0111] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A single cell analysis method, characterized in that: Using a microporous chip, the microporous chip has a plurality of double-pass penetrating micropores, and the micropores can perform capillary action, including the following steps: S1. After a single cell suspension or a single cell nucleus suspension having a first section of cell identity tags introduced into an in-situ labeled object of a single cell or a single cell nucleus of a tissue sample to be tested is mixed with microbeads with nucleic acid molecules of a known base sequence, the mixture is loaded into a micropore of a micropore chip under capillary action, and the second section of cell identity tags on the microbeads is combined with the nucleic acid of the single cell or the single cell nucleus, wherein the nucleic acid molecules of a known base sequence on the microbeads include conditionally cleavable sites, wherein the conditionally cleavable sites include any one of dU base modification, disulfide bond modification, photocleavable linker, and restriction endonuclease recognition sequence; S2. Performing nucleic acid molecule polymerization and amplification on the microporous chip in S1 to construct a single-cell sequencing library.

2. The single cell analysis method according to claim 1, characterized in that Step S1 also includes the following step: using a fixative to fix the single cells or cell nuclei in the single cell suspension or single cell nucleus suspension.

3. The single cell analysis method according to claim 1, characterized in that In step S1, when the nucleic acid molecule with known base sequence on the microbead is hybridized with the single cell or single cell nucleus, the in situ labeled object in the single cell or single cell nucleus is RNA or DNA, and correspondingly, the nucleic acid molecule with known base sequence on the microbead includes nucleic acid; Alternatively, the in situ labeling object in a single cell or a single cell nucleus is a protein, and correspondingly, the nucleic acid molecules with known base sequences on the microbeads include nucleic acids modified on antibodies.

4. The single cell analysis method according to claim 3, characterized in that When the in situ labeling object in a single cell or a single cell nucleus is RNA or protein, the nucleic acid molecule with a known base sequence on the microbead is a single-stranded oligonucleotide with a known base sequence; when the in situ labeling object in a single cell or a single cell nucleus is DNA, the nucleic acid molecule with a known base sequence on the microbead is a transposase adapter sequence with a known base sequence.

5. The single cell analysis method according to any one of claims 1 to 4, characterized in that: The microbeads are directly coupled to nucleic acid molecules of known base sequences in any of the following ways: condensation acylation reaction based on carboxyl amino groups, cross-linking reaction based on biotin and streptavidin, ester bond connection, disulfide bond connection, and dipole interaction.

6. The single cell analysis method according to any one of claims 1 to 4, characterized in that: In step S1, when the single cell suspension or single cell nucleus suspension of the tissue sample to be tested is mixed with microbeads with nucleic acid molecules of known base sequence and then loaded into the micropores of the microporous chip, the following steps are included: the single cell suspension or single cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence are adsorbed into each of the micropores along with the liquid on the surface of the microporous chip through capillary action, wherein each of the micropores can accommodate multiple microbeads.

7. The single cell analysis method according to any one of claims 1 to 4, characterized in that: In step S1, a single cell nucleus suspension of the tissue sample to be tested is obtained by the following steps: a cell line of the sample to be tested is subjected to a lysis reaction by a lysis solution, and the lysis is terminated after a predetermined lysis time to obtain a single cell nucleus of the sample to be tested.

8. The single cell analysis method according to any one of claims 1 to 4, characterized in that: In step S2, after the single cell suspension or single cell nucleus suspension of the tissue sample to be tested and the microbeads with nucleic acid molecules of known base sequence are loaded onto the chip, the evaporation of the liquid on the surface of the microporous chip is promoted by natural evaporation or vacuum evaporation.

9. The single cell analysis method according to any one of claims 1 to 4, characterized in that: In step S2, when the nucleic acid molecule polymerization and amplification are performed on the microporous chip in S1, the following steps are included: the microporous chip is sealed with sealing oil, and the in situ labeled object in the single cell or single cell nucleus is released by constant temperature heating to hybridize with the nucleic acid molecule of known base sequence on the microbead.

10. The single cell analysis method according to any one of claims 1 to 4, characterized in that: The single-cell sequencing library includes single-cell transcriptome data and / or chromatin accessibility genomic data.

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