Method and kit for high-throughput tagging of cell nucleic acid molecules

Abstract

A method of introducing a dual cell-specific tag in nucleic acid molecules derived from cells, and the construction of a library of nucleic acid molecules for single-cell transcriptome sequencing, single-cell chromatin accessibility sequencing or multi-omic single-cell transcriptome + chromatin accessibility sequencing on the basis of the method, or a method for performing high-throughput sequencing on single-cell transcriptome, single-cell chromatin accessibility or multi-omic single cell transcriptome + chromatin accessibility. Further provided are a library of nucleic acid molecules constructed using the method, and a kit for implementing the method.

Description

Method and kit for high-throughput labeling of cellular nucleic acid molecules Technical Field

[0001] This application relates to the field of high-throughput single-cell omics, in particular, high-throughput single-cell transcriptome sequencing technology, high-throughput single-cell chromatin accessibility (ATAC, Assay for Transposase-Accessible Chromatin) sequencing technology, and high-throughput single-cell transcriptome + chromatin accessibility multi-omics sequencing technology. Background Art

[0002] The development of single-cell omics sequencing technology has significantly deepened our understanding of cellular diversity and heterogeneity, revolutionizing research in diverse biological and biomedical fields, including developmental biology, cancer and other diseases, assisted reproduction, immunology, neuroscience, and microbiology. Existing single-cell sequencing technologies primarily include single-cell genome sequencing, transcriptome sequencing, methylation sequencing, chromatin accessibility sequencing, and single-cell multi-omics sequencing that encompasses all of these omics. Essentially, single-cell sequencing analyzes the sequence, copy number, modification status, and interactions of DNA and RNA within individual cells, revealing genomic variations in the genome, transcriptome, methylation status, and chromatin accessibility. To more precisely characterize cellular heterogeneity across samples, analyze single-cell gene regulatory networks, and paint a comprehensive cellular picture, there remains a pressing need for single-cell sequencing technologies with higher throughput, more modalities, better data quality, and lower costs.

[0003] High-throughput single-cell library construction currently primarily involves barcoding cells in microfluidic droplets or microplates. However, all current commercial single-cell library construction technologies based on microfluidic droplets and microplates, such as 10x Genomics (Zheng GX, et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun. 2017 Jan 16; 8:14049. doi:10.1038 / ncomms14049. PMID:28091601), have the following drawbacks: low cell throughput, high library construction costs, high microreactor emptying rates, and high rates of pseudo-single cells.

[0004] In summary, it is very necessary to construct a technical method that is simple to operate, low-cost, has high cell throughput, reliable data quality, and is also suitable for multi-omics single-cell library construction.

[0005] Summary of the Invention

[0006] To address the issues of high-throughput single-cell library construction technology based on microreaction (e.g., water-in-oil droplets, nanopores) cell barcode labeling and the inability of existing high-throughput library construction technologies based on microfluidic droplets and combinatorial labeling to simultaneously achieve the following: high cell throughput, simple operation, low price, low false single cell rate, low microreaction system empty rate, good data quality, and strong applicability, the inventors of this application have developed a new and improved library construction technology solution based on the existing mature microreaction (e.g., water-in-oil droplets, nanopores) cell barcode labeling platform.

[0007] Marking method

[0008] Therefore, in a first aspect, the present application provides a method for labeling nucleic acid molecules from cells or cell nuclei, comprising the following steps:

[0009] (1) providing a plurality of fixed and permeabilized cells or cell nuclei, wherein the cells or cell nuclei contain nucleic acid molecules to be labeled; and,

[0010] a plurality of beads coupled to a plurality of first oligonucleotide molecules, wherein the first oligonucleotide molecules contain a first tag sequence;

[0011] Furthermore, the plurality of first oligonucleotide molecules on the same bead have the same first tag sequence, and the first oligonucleotide molecules on different beads have different first tag sequences;

[0012] (2) randomly distributing the plurality of beads and the plurality of cells or cell nuclei into different first discrete partitions, releasing the first oligonucleotide molecule from the beads and contacting the first oligonucleotide molecule with the cells or cell nuclei within the first discrete partitions, thereby generating a first nucleic acid molecule derived from the nucleic acid molecule to be labeled within the cells or cell nuclei, wherein the first nucleic acid molecule contains the first tag sequence or its complementary sequence;

[0013] (3) mixing the cells or cell nuclei containing the first nucleic acid molecule from different first discrete partitions and redistributing them into different second discrete partitions;

[0014] (4) contacting a second oligonucleotide molecule containing a second tag sequence with the first nucleic acid molecule within the second discrete partition to generate a second nucleic acid molecule containing the first tag sequence or its complementary sequence and the second tag sequence or its complementary sequence;

[0015] wherein the second oligonucleotide molecules in the same second discrete partition have the same second tag sequence, and the second oligonucleotide molecules in different second discrete partitions have different second tag sequences;

[0016] The cells are naturally occurring cells or recombinant cells, or a mixture of the two; the cell nuclei are cell nuclei derived from naturally occurring cells or recombinant cells, or a mixture of the two.

[0017] In certain embodiments, the recombinant cell refers to a cell comprising a modified (e.g., artificially modified) nucleic acid molecule (e.g., gene) and / or its product (e.g., protein, RNA), wherein the modification includes but is not limited to increasing or decreasing the copy number of the endogenous gene of the cell, mutating the endogenous gene of the cell, upregulating, downregulating or silencing the expression of the endogenous gene product of the cell, introducing an exogenous nucleic acid molecule into the cell (the exogenous nucleic acid molecule is integrated into the genome of the cell or exists in a non-integrated form), etc.

[0018] The method of the present application can be used to label unmodified nucleic acid molecules in the cell / cell nucleus, and can also be used to label modified nucleic acid molecules in the cell / cell nucleus (for example, modified endogenous nucleic acid molecules (for example, genes) of the cell or introduced exogenous nucleic acid molecules contained in the cell).

[0019] In certain embodiments, in step (3), the 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 At least 10 8 2-10 pieces, 2-10 pieces 2 2-10 3 2-10 4 2-10 5 2-10 6 2-10 7 2-10 8 or 2-10 9 The cells or cell nuclei containing the first nucleic acid molecule in each of the first discrete partitions are mixed and redistributed into different second discrete partitions.

[0020] It is easy for a person skilled in the art to understand that, since the first tag sequence contained in the first oligonucleotide molecule is specific to the first discrete partition, all the first nucleic acid molecules derived from cells or cell nuclei in the same first discrete partition in step (2) contain the same first tag sequence or its complementary sequence. The second tag sequence contained in the second oligonucleotide molecule is specific to the second discrete partition, and therefore, all the second nucleic acid molecules derived from cells or cell nuclei assigned to the same second discrete partition in step (4) contain the same second tag sequence or its complementary sequence. Thus, the first tag sequence and the second tag sequence can be used together to identify the cell from which the sequencing data originated.

[0021] It is easy to understand that the labeling method provided in this application can be used for high-throughput library construction and sequencing of single-cell omics. Therefore, in step (1), the cells or cell nuclei can be of the same source or a mixture of different sources; the cells or cell nuclei can be derived from the same cell line or from different cell lines, from the same tissue or from different tissues, from the same individual or from different individuals, from the same species or from different species. The cells or cell nuclei can also be a mixture of cells and cell nuclei.

[0022] In certain embodiments, a single said first discrete partition contains one said bead.

[0023] In certain embodiments, each of the first discrete partitions independently contains one or more cells or cell nuclei.

[0024] In certain embodiments, each of the first discrete partitions independently contains 0-10 (e.g., 0-2, 0-3, 0-4, 0-5, 0-8, 1-2, 1-3, 1-4, 1-5, 1-8, 1-10, 2-3, 2-4, 2-5, 2-8, 2-10, 3-4, 3-5, 3-8, 3-10, 4-5, 4-8, 4-10) cells or nuclei.

[0025] In certain embodiments, each of the second discrete partitions independently contains one or more cells or cell nuclei derived from the first discrete partition that contain the first nucleic acid molecule.

[0026] In certain embodiments, each of the second discrete partitions independently contains 0-10 7 (e.g., 0-10, 0-10 2 0-10 3 0-10 4 0-105 0-10 6 0-10 7 pcs, 1-10 pcs, 1-10 2 1-10 3 1-10 4 1-10 5 1-10 6 or 1-10 7 ) cells or cell nuclei derived from the first discrete partition that contain the first nucleic acid molecule.

[0027] In certain embodiments, in step (2), the method randomly distributes the plurality of beads and the plurality of cells or cell nuclei into different first discrete partitions by a microdroplet microfluidics system or a microplate system.

[0028] In certain embodiments, the droplet microfluidic system is selected from but not limited to: a microfluidic oil-in-water system of a 10X GENOMICS platform, a microfluidic system of a Fluidigm C1 platform, and a microfluidic system of a Biorad ddSEQ system.

[0029] In certain embodiments, the microplate system is selected from but not limited to: a microplate system of a BD Rhapsody platform and a microplate system of a Neocell platform.

[0030] In certain embodiments, the method uses methanol to fix and permeabilize the cells, or alternatively, uses formaldehyde or paraformaldehyde and Triton X-100 to fix and permeabilize the cells.

[0031] In certain embodiments, the method comprises fixing and permeabilizing the cells by a treatment selected from the group consisting of:

[0032] (i) treating the cells with 60%-100% (e.g., 60%-80%, 70%-80%, 70%-85%, 70%-90%, 75%-80%, 75%-85%, 75%-90%, 75%-100%, 80%-85%, 80%-90%, 80%-100%, or 80%) methanol at -40°C to -10°C (e.g., -25°C to -15°C or -20°C) for 5-30 min (e.g., 8-20 min or 10 min) to fix and permeabilize the cells;

[0033] or,

[0034] (ii) (a) fixing the cells by treating the cells with formaldehyde or paraformaldehyde at a concentration of 0.05% to 5% (e.g., 0.5% to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, or 1%) at 0°C to 37°C (e.g., 15°C to 30°C or 25°C) for 5-30 minutes (e.g., 5-20 minutes or 10 minutes); and,

[0035] (b) using Triton at a concentration of 0.05% to 2% (e.g., 0.05% to 0.2%, 0.05% to 0.25%, 0.05% to 0.3%, 0.05% to 0.5%, 0.05% to 0.8%, 0.05% to 1%, 0.1% to 0.2%, 0.1% to 0.25%, 0.1% to 0.3%, 0.1% to 0.4%, 0.1% to 0.5%, 0.1% to 0.8%, 0.1% to 1%, 0.2% to 0.25%, 0.2% to 0.3%, 0.2% to 0.4%, 0.2% to 0.5%, 0.2% to 0.8%, 0.2% to 1%, or 0.2%) Treat the cells with X-100 at -4°C to 10°C (eg, 0°C to 4°C) for 0.5-10 min (eg, 1-5 min or 3 min) to permeabilize the cells.

[0036] In certain embodiments, cells are fixed and permeabilized by treating the cells with 80% methanol at -20°C for 10 minutes.

[0037] In certain embodiments, the methods use formaldehyde or paraformaldehyde and digitonin to fix and permeabilize the cell nuclei.

[0038] In certain embodiments, the method further comprises administering IGEPAL (e.g., The cell nuclei were permeabilized with CA-630) and / or Tween-20.

[0039] In certain embodiments, the method comprises fixing and permeabilizing the cell nuclei by a treatment selected from the group consisting of:

[0040] (i) fixing the cell nucleus, wherein the fixing method is selected from:

[0041] (a) fixing the cell nuclei by treating the cell nuclei with formaldehyde at a concentration of 0.05%-4% (e.g., 0.5%-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, or 1%) at 0°C to 30°C (e.g., 15°C to 28°C or 25°C) for 2-20 minutes (e.g., 5-15 minutes or 10 minutes); or,

[0042] (b) fixing the cell nuclei by treating the cell nuclei with paraformaldehyde at a concentration of 0.05% to 4% (e.g., 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, or 1.6%) at 0° C. to 30° C. (e.g., 15° C. to 28° C. or 25° C.) for 1 to 15 minutes (e.g., 1 to 10 minutes or 5 minutes);

[0043] as well as,

[0044] (ii) permeabilizing the cell nuclei by treating the cell nuclei with a permeabilization solution containing digitonin at -4°C to 10°C (eg, 0°C to 4°C) for 0.5-10 min (eg, 1-5 min or 3 min).

[0045] In certain embodiments, the permeabilization solution further comprises IGEPAL (e.g., CA-630) and / or Tween-20.

[0046] In certain embodiments, the concentration of digitonin in the permeabilization solution is 0.0005%-0.05% (eg, 0.0008%-0.005%, 0.0005%-0.002%, 0.0008%-0.002%, or 0.001%).

[0047] In certain embodiments, the permeabilization solution contains IGEPAL (e.g., CA-630) at a concentration of 0.005%-0.1% (e.g., 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0048] In certain embodiments, the concentration of Tween-20 in the permeabilization solution is 0.005%-0.1% (eg, 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0049] In certain embodiments, the cell nuclei are fixed by treating the cell nuclei with 1% formaldehyde for 10 min at room temperature, and after fixation, the cell nuclei are fixed with a solution containing 0.001% digitonin, 0.01% IGEPAL (e.g., The cell nuclei are permeabilized by treating the cell nuclei with a permeabilization solution containing CA-630) and 0.01% Tween-20 at 0° C. to 4° C. for 2-4 min (e.g., 3 min).

[0050] In certain embodiments, the method of labeling nucleic acid molecules from cells or cell nuclei comprises one or more selected from the following:

[0051] (1) In step (1), at least 2 (e.g., at least 10, at least 10 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 2-10 pieces, 2-10 pieces 2 2-10 3 2-10 4 2-10 5 2-10 6 2-10 7 2-10 8 or 2-10 9 ) cells or cell nuclei; and / or, providing at least 2 (e.g., at least 10, at least 10 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 At least 10 8 2-10 pieces, 2-10 pieces 2 2-10 3 2-10 4 2-10 5 2-10 6 2-10 7 2-10 8 or 2-10 9 individual) beads;

[0052] (2) the first discrete partitions are discrete micropores or discrete microdroplets (e.g., water-in-oil droplets);

[0053] (3) The beads are coupled to at least 2 (e.g., at least 10, at least 10 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 At least 10 8 2-10 pieces, 2-10 pieces 2 2-10 3 2-10 4 2-105 2-10 6 2-10 7 2-10 8 or 2-10 9 ) the first oligonucleotide molecule;

[0054] (4) the bead is capable of releasing the first oligonucleotide molecule spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a specific chemical or phase, exposure to light, a reducing agent, etc.);

[0055] (5) The beads are gel beads;

[0056] (6) In step (3), the cells or cell nuclei are divided into at least 2 (e.g., at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 20, at least 24, at least 50, at least 96, at least 100, at least 200, at least 384, at least 400, 2-5, 2-10, 2-50, 2-80, 2-100, 2-500, 2-10 3 2-10 4 2-10 5 or 2-10 6 ) the second discrete partitions, wherein each of the second discrete partitions contains at least one cell or cell nucleus;

[0057] (7) The second discrete partitions are discrete wells in a porous plate;

[0058] (8) After step (3) and before step (4), the method further includes the steps of lysing cells and / or purifying the first nucleic acid molecule.

[0059] Furthermore, the labeling methods of the present invention are respectively:

[0060] The nucleic acid molecule to be labeled is mRNA:

[0061] In certain embodiments, the nucleic acid molecule to be labeled is mRNA, and the first oligonucleotide molecule is first oligonucleotide molecule a.

[0062] In certain embodiments, step (2) comprises the following steps:

[0063] (i) (a) reverse transcribing the nucleic acid molecule to be labeled with the first oligonucleotide molecule a within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the nucleic acid molecule to be labeled, formed using the first oligonucleotide molecule a as a reverse transcription primer, and a 3'-terminal overhang; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A to the cDNA chain generated in (a) and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the primer A comprises, from the 5' end to the 3' end, a consensus sequence O and a complementary sequence to the 3'-terminal overhang;

[0064] or,

[0065] (ii) (a) reverse transcribing the nucleic acid molecule to be labeled using primer B within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the nucleic acid molecule to be labeled formed using primer B as a reverse transcription primer, and a 3'-terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; and, (b) annealing the first oligonucleotide molecule a with the cDNA chain generated in (a) within the first discrete partition, and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3'-terminal overhang.

[0066] Various suitable methods can be used to form or add an overhang to the 3' end of a cDNA strand. In certain embodiments, an overhang can be formed or added to the 3' end of a cDNA strand by using a reverse transcriptase having terminal transfer activity.

[0067] In certain embodiments, in said step (ii), step (ii)(a) is performed before or after partitioning said cells or cell nuclei into said first discrete partitions.

[0068] In certain embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and multiple first oligonucleotide molecules a coupled to the same bead have different unique molecular tag sequences. In certain embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof.

[0069] In certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T.

[0070] In certain embodiments, the 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0071] In certain embodiments, step (4) comprises the following steps:

[0072] In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer C as primers, and the generated extension product is the second nucleic acid molecule;

[0073] Wherein, the second oligonucleotide molecule comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof.

[0074] The nucleic acid molecule to be labeled is genomic DNA:

[0075] In certain embodiments, the nucleic acid molecule to be labeled is genomic DNA, and the first oligonucleotide molecule is the first oligonucleotide molecule b.

[0076] In certain embodiments, step (2) comprises the following steps:

[0077] (a) incubating the nucleic acid molecule to be labeled with a transposase complex I; wherein the transposase complex I contains a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a first transferred strand and a second transferred strand, the first transferred strand comprising a transposase recognition sequence and a consensus sequence R2 or a partial sequence thereof, and the second transferred strand comprising a transposase recognition sequence and a consensus sequence R1 or a partial sequence thereof; and the incubation is performed under conditions that allow the nucleic acid molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strands to be ligated to the ends of the nucleic acid fragments (e.g., the 5' ends of the nucleic acid fragments); thereby generating double-stranded nucleic acid fragments whose 5' ends respectively contain the consensus sequence R2 or a partial sequence thereof and the consensus sequence R1 or a partial sequence thereof; and,

[0078] (b) Within the first discrete partition, the first oligonucleotide molecule b is connected to the double-stranded nucleic acid fragment generated in (a) (for example, by using a nuclease), and an extension reaction is performed to generate an extension product, which is the first nucleic acid molecule; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof and the first tag sequence.

[0079] In certain embodiments, the first nucleic acid molecule comprises a sequence derived from a genomic DNA fragment in an open chromatin region in the cell or cell nucleus.

[0080] In certain embodiments, step (a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0081] In certain embodiments, the 5' end of the consensus sequence R1 of the transposase complex I or a partial sequence thereof is phosphorylated.

[0082] In certain embodiments, step (4) comprises the following steps:

[0083] In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer D as primers, and the generated extension product is the second nucleic acid molecule;

[0084] The second oligonucleotide molecule comprises, from the 5' end to the 3' end: the consensus sequence P2 or a partial sequence thereof, the second tag sequence, the consensus sequence R2 or a partial sequence thereof; and the primer D comprises the consensus sequence P1 or a partial sequence thereof.

[0085] The nucleic acid molecules to be labeled are mRNA and genomic DNA from the same cell:

[0086] In certain embodiments, the nucleic acid molecules to be labeled are mRNA and genomic DNA, and the mRNA and genomic DNA have the same cell origin;

[0087] Furthermore, the first oligonucleotide molecule includes a first oligonucleotide molecule a and a first oligonucleotide molecule b, and the second oligonucleotide molecule includes a second oligonucleotide molecule a and a second oligonucleotide molecule b;

[0088] The beads are coupled to a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b at the same time; and the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence.

[0089] In certain embodiments, step (2) comprises the following steps:

[0090] (A)(i)(a) reverse transcribing the mRNA molecule to be labeled with the first oligonucleotide molecule a within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled, formed using the first oligonucleotide molecule a as a reverse transcription primer, and a 3'-terminal overhang; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A to the cDNA chain generated in (a) and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the primer A comprises, from the 5' end to the 3' end, a consensus sequence O and a complementary sequence to the 3'-terminal overhang;

[0091] or,

[0092] (ii) (a) reverse transcribing the mRNA molecule to be labeled with primer B within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled, formed using primer B as a reverse transcription primer, and a 3'-terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; and, (b) annealing the first oligonucleotide molecule a with the cDNA chain generated in (a) within the first discrete partition, and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3'-terminal overhang;

[0093] and,

[0094] (B)(a) incubating the DNA molecule to be labeled with a transposase complex I; wherein the transposase complex I is as defined in claim 9; and the incubation is performed under conditions that allow the DNA molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strands to be ligated to the ends of the nucleic acid fragments (e.g., the 5' ends of the nucleic acid fragments); thereby generating double-stranded nucleic acid fragments whose 5' ends contain a consensus sequence R2 or a partial sequence thereof and a consensus sequence R1 or a partial sequence thereof, respectively; and,

[0095] (b) within the same first discrete partition as (A), ligating the first oligonucleotide molecule b to the double-stranded nucleic acid fragment generated in (a), and performing an extension reaction to generate an extension product, which is the first nucleic acid molecule b; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0096] Wherein, the step (A) and the step (B) can be performed in any order (for example, (A) first and then (B), (B) first and then (A), or simultaneously).

[0097] In certain embodiments, in step (A)(ii), step (A)(ii)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0098] In certain embodiments, step (B)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0099] In certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T.

[0100] In certain embodiments, the first nucleic acid molecule b comprises a sequence derived from a genomic DNA fragment in an open chromatin region in the cell or cell nucleus.

[0101] In certain embodiments, the 5' end of the consensus sequence R1 or a partial sequence thereof in the transposase complex I is phosphorylated.

[0102] In certain embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and multiple first oligonucleotide molecules a coupled to the same bead have different unique molecular tag sequences. In certain embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof.

[0103] In certain embodiments, the 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0104] In certain embodiments, step (4) comprises the following steps:

[0105] (a) amplifying the first nucleic acid molecule a in the second discrete partition using the second oligonucleotide molecule a and primer C as primers, whereby the generated extension product is the second nucleic acid molecule a;

[0106] Wherein, the second oligonucleotide molecule a comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof;

[0107] and

[0108] (b) amplifying the first nucleic acid molecule b within the same second discrete partition using the second oligonucleotide molecule b and primer D as primers, whereby the generated extension product is the second nucleic acid molecule b;

[0109] Wherein, the second oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P2 or a partial sequence thereof, the second tag sequence, the consensus sequence R2 or a partial sequence thereof; the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0110] Wherein, the step (a) and the step (b) can be performed in any order (for example, (a) first and then (b), (b) first and then (a), or simultaneously).

[0111] Furthermore, the library construction methods of the present invention are respectively:

[0112] In a second aspect, the present application also provides a method for constructing a nucleic acid molecule library, which comprises:

[0113] (1) generating a plurality of labeled second nucleic acid molecules according to the method according to any one of the first aspects, and,

[0114] (2) recovering and / or combining a plurality of said second nucleic acid molecules,

[0115] Thus, a nucleic acid molecule library is obtained.

[0116] In certain embodiments, in step (2), the second nucleic acid molecules generated in a plurality of the second discrete partitions are recovered and / or pooled.

[0117] The nucleic acid molecule to be labeled is mRNA:

[0118] In certain embodiments, the method comprises:

[0119] (a) generating a plurality of labeled second nucleic acid molecules according to the method described in the section "The nucleic acid molecule to be labeled is mRNA" of the first aspect above,

[0120] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions; and

[0121] (c) randomly fragmenting the second nucleic acid molecule and adding a linker sequence;

[0122] Thus, the sequence of the nucleic acid molecule library is obtained.

[0123] In certain embodiments, the cell is a T cell or a B cell.

[0124] In certain embodiments, the method further comprises, after step (a) and before step (c), a step of enriching the target nucleic acid molecule; the target nucleic acid molecule is a second nucleic acid molecule comprising: (i) a nucleotide sequence encoding a T cell receptor (TCR) or a B cell receptor (BCR) or a partial sequence thereof (e.g., a V(D)J sequence), and / or (ii) a complementary sequence to (i).

[0125] In certain embodiments, in step (c), the second nucleic acid molecule is randomly fragmented by a transposase and an adapter sequence is added to the 5' end thereof.

[0126] In certain embodiments, the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.

[0127] In certain embodiments, the method further comprises step (d):

[0128] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c).

[0129] In certain embodiments, step (d) comprises: amplifying the product of step (c) using primer E and primer F, wherein primer E comprises a consensus sequence P1 and an optional third tag sequence, and primer F comprises from 5' to 3': a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, a consensus sequence R2 or a partial sequence thereof.

[0130] The nucleic acid molecule to be labeled is genomic DNA:

[0131] In certain embodiments, the method comprises:

[0132] (a) generating a plurality of labeled second nucleic acid molecules according to the method described in the section "The nucleic acid molecule to be labeled is genomic DNA" of the first aspect above, and,

[0133] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions;

[0134] Thus, the nucleic acid molecule library sequence is obtained.

[0135] In certain embodiments, the method further comprises step (c):

[0136] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

[0137] In certain embodiments, step (c) comprises amplifying the product of step (b) using primer E' and primer F', wherein primer E' comprises a consensus sequence P1 and an optional third tag sequence, and primer F' comprises a consensus sequence P2 and an optional fourth tag sequence.

[0138] The nucleic acid molecules to be labeled are mRNA and genomic DNA from the same cell:

[0139] In certain embodiments, the method comprises:

[0140] (a) generating a plurality of labeled second nucleic acid molecules according to the method described in the section "The nucleic acid molecules to be labeled are mRNA and genomic DNA from the same cell" of the first aspect above, comprising a plurality of second nucleic acid molecules a and a plurality of second nucleic acid molecules b, and,

[0141] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions;

[0142] Thus, the nucleic acid molecule library sequence is obtained.

[0143] In certain embodiments, the method further comprises, after step (b), step (c): randomly fragmenting the second nucleic acid molecule a and adding a linker sequence.

[0144] In certain embodiments, in step (c), the second nucleic acid molecule a is randomly fragmented by a transposase and a linker sequence is added to the 5' end thereof.

[0145] In certain embodiments, the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.

[0146] In another embodiment, before step (c), the method further comprises specifically enriching the second nucleic acid molecule a from the product of step (b).

[0147] In certain embodiments, the method specifically amplifies and enriches the second nucleic acid molecule a by using a primer G carrying a biotin label.

[0148] In certain embodiments, the primer G contains the consensus sequence O or a partial sequence thereof, or the primer G contains the consensus sequence T or a partial sequence thereof.

[0149] In certain embodiments, the amplification and enrichment further comprises using a primer H, wherein the primer H comprises a consensus sequence P1 or a partial sequence thereof.

[0150] In certain embodiments, the method further comprises step (d):

[0151] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c).

[0152] In certain embodiments, step (c) comprises: amplifying the product of step (c) using primer E and primer F, wherein primer E comprises a consensus sequence P1 and an optional third tag sequence, and primer F comprises from 5' to 3': a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, and a consensus sequence R2.

[0153] In certain embodiments, the method further comprises step (d)':

[0154] A step of purifying and / or amplifying the second nucleic acid molecule b containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

[0155] In certain embodiments, step (d)' comprises: amplifying the product of step (b) using primer E' and primer F', wherein primer E' comprises a consensus sequence P1 and an optional third tag sequence, and primer F' comprises a consensus sequence P2 and an optional fourth tag sequence.

[0156] In a third aspect, the present application further provides a method for performing omics sequencing on a cell or a cell nucleus, comprising:

[0157] Constructing a nucleic acid molecule library according to any one of the methods described in the second aspect above; and,

[0158] The nucleic acid molecule library is sequenced.

[0159] In certain embodiments, before sequencing, at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, 2-5, 2-10, 2-20, 2-30, 2-40 or 2-50 nucleic acid molecule libraries are combined and then sequenced; wherein each nucleic acid molecule library each has multiple nucleic acid molecules (i.e., amplification products), and the multiple nucleic acid molecules in the same library have the same third tag sequence or the same fourth tag sequence; and nucleic acid molecules derived from different libraries have different third tag sequences or different fourth tag sequences from each other.

[0160] In a fourth aspect, the present application further provides a nucleic acid molecule library constructed by the method described in any one of the second aspects above.

[0161] product

[0162] In a fifth aspect, the present application further provides a reagent composition having characteristics selected from I, II and III:

[0163] (I) The reagent composition comprises a second oligonucleotide molecule a, wherein the sequence of the second oligonucleotide molecule a comprises, from the 5′ end to the 3′ end, a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof;

[0164] Furthermore, the reagent composition further comprises one or more selected from the following:

[0165] (Ia) a plurality of beads coupled with a plurality of first oligonucleotide molecules a, wherein the first oligonucleotide molecules a contain a first tag sequence;

[0166] Furthermore, the plurality of first oligonucleotide molecules a on the same bead have the same first tag sequence, and the first oligonucleotide molecules a on different beads have different first tag sequences;

[0167] In certain embodiments, the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; or (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3' end overhang of the cDNA;

[0168] In some embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in some embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof;

[0169] (Ib) primer A or primer B, wherein the primer A comprises a consensus sequence O and a complementary sequence to the 3'-end overhang of the cDNA from the 5' end to the 3' end, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0170] (Ic) primer C, wherein the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0171] (Id) primer E and / or primer F, wherein the primer E comprises a consensus sequence P1 and an optional third tag sequence, and the primer F comprises, from 5' to 3', a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, a consensus sequence R2 or a partial sequence thereof;

[0172] (Ie) a transposase complex II, comprising a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break a double-stranded nucleic acid; wherein the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;

[0173] In certain embodiments, the cDNA 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang);

[0174] (II) the reagent composition comprises a second oligonucleotide molecule b, wherein the sequence of the second oligonucleotide molecule b comprises, from the 5′ end to the 3′ end, a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof;

[0175] Furthermore, the reagent composition further comprises one or more selected from the following:

[0176] (II-a) a plurality of beads coupled with a plurality of first oligonucleotide molecules b, wherein the first oligonucleotide molecules b contain a first tag sequence;

[0177] Furthermore, the plurality of first oligonucleotide molecules b on the same bead have the same first tag sequence, and the first oligonucleotide molecules b on different beads have different first tag sequences;

[0178] In certain embodiments, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0179] (II-b) transposase complex I, wherein the transposase complex I is as defined in the section "the nucleic acid molecule to be labeled is genomic DNA" in the labeling method of the first aspect;

[0180] (II-c) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0181] (II-d) primer E' and / or primer F', wherein the primer E' comprises a consensus sequence P1 and an optional third tag sequence, and the primer F' comprises a consensus sequence P2 and an optional fourth tag sequence;

[0182] (III) The reagent composition comprises a second oligonucleotide molecule a and a second oligonucleotide molecule b; wherein the sequence of the second oligonucleotide molecule a comprises, from the 5' end to the 3' end, a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof; and the sequence of the second oligonucleotide molecule b comprises, from the 5' end to the 3' end, a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof;

[0183] Furthermore, the reagent composition further comprises one or more selected from the following:

[0184] (III-a) a plurality of beads to which a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b are simultaneously coupled, wherein the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence, the first oligonucleotide molecules a on different beads have different first tag sequences, and the first oligonucleotide molecules b on different beads have different first tag sequences;

[0185] In certain embodiments, the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; or, (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3' end overhang of the cDNA; and / or, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0186] In some embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in some embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof;

[0187] (III-b) Primer A or primer B, wherein the primer A comprises a consensus sequence O and a complementary sequence to the 3'-end overhang of the cDNA from its 5' end to its 3' end, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from its 5' end to its 3' end; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0188] (III-c) transposase complex I, wherein the transposase complex I is as defined in the section "the nucleic acid molecule to be labeled is genomic DNA" in the labeling method of the first aspect;

[0189] (III-d) primer C, the primer C comprising the consensus sequence O or a partial sequence thereof, or the primer C comprising the consensus sequence T or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0190] (III-e) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0191] (III-f) primer E and / or primer F, wherein the primer E comprises the consensus sequence P1 and an optional third tag sequence, and the primer F comprises, from 5' to 3', the consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, and the consensus sequence R2 or a partial sequence thereof;

[0192] (III-g) primer E' and / or primer F', wherein the primer E' comprises a consensus sequence P1 and an optional third tag sequence, and the primer F' comprises a consensus sequence P2 and an optional fourth tag sequence;

[0193] (III-h) comprises primer G and / or primer H, wherein primer G carries a biotin label and contains a consensus sequence O or a partial sequence thereof or a consensus sequence T or a partial sequence thereof, and primer H contains a consensus sequence P1 or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0194] (III-i) a transposase complex II, comprising a transposase and a transposition sequence that the transposase can recognize and bind to, and is capable of cleaving or breaking a double-stranded nucleic acid; wherein the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; and in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;

[0195] In certain embodiments, the cDNA 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0196] In certain embodiments, the reagent composition further comprises a reagent for fixing and / or permeabilizing cells or cell nuclei.

[0197] In certain embodiments, the reagent composition further comprises methanol, formaldehyde and / or paraformaldehyde.

[0198] In certain embodiments, the reagent composition further comprises Triton X-100, digitonin, IGEPAL (e.g., CA-630), and / or Tween-20.

[0199] In certain embodiments, the reagent composition further comprises: an RNase inhibitor, mineral oil, a buffer, dNTPs, one or more nucleic acid polymerases (e.g., DNA polymerases; e.g., DNA polymerases having strand displacement activity and / or high fidelity), reagents for recovering or purifying nucleic acids (e.g., magnetic beads), a well plate, or any combination thereof.

[0200] In certain embodiments, the reagent composition further comprises reagents for sequencing, such as reagents for next-generation sequencing.

[0201] In a sixth aspect, the present application further provides a kit comprising: a multi-reaction system comprising a plurality of oligonucleotide molecules, each of the oligonucleotide molecules comprising a specific tag sequence;

[0202] Furthermore, in the multi-reaction system, the oligonucleotide molecules in each reaction system have the same tag sequence, and the oligonucleotide molecules in different reaction systems have different tag sequences.

[0203] In certain embodiments, the oligonucleotide molecule further comprises a consensus sequence P1 or a partial sequence thereof, or the oligonucleotide molecule further comprises a consensus sequence P2 or a partial sequence thereof.

[0204] In certain embodiments, the multi-reaction system comprises at least 2 (e.g., at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 20, at least 24, at least 50, at least 96, at least 100, at least 200, at least 384, at least 400, 2-5, 2-10, 2-50, 2-80, 2-100, 2-500, 2-10 3 2-10 4 2-10 5 2-10 6 ) a multi-reaction system containing oligonucleotides;

[0205] The multi-reaction system is preferably a multi-well plate, and the oligonucleotides can be free or immobilized in the reaction system.

[0206] In a seventh aspect, the present application provides a method for fixing and permeabilizing cells, comprising the following steps:

[0207] (i) treating the cells with 60%-100% (e.g., 60%-80%, 70%-80%, 70%-85%, 70%-90%, 75%-80%, 75%-85%, 75%-90%, 75%-100%, 80%-85%, 80%-90%, 80%-100%, or 80%) methanol at -40°C to -10°C (e.g., -25°C to -15°C or -20°C) for 5-30 min (e.g., 8-20 min or 10 min) to fix and permeabilize the cells;

[0208] or,

[0209] (ii) (a) fixing the cells by treating the cells with formaldehyde or paraformaldehyde at a concentration of 0.05% to 5% (e.g., 0.5% to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, or 1%) at 0°C to 37°C (e.g., 15°C to 30°C or 25°C) for 5-30 minutes (e.g., 5-20 minutes or 10 minutes); and,

[0210] (b) using Triton at a concentration of 0.05% to 2% (e.g., 0.05% to 0.2%, 0.05% to 0.25%, 0.05% to 0.3%, 0.05% to 0.5%, 0.05% to 0.8%, 0.05% to 1%, 0.1% to 0.2%, 0.1% to 0.25%, 0.1% to 0.3%, 0.1% to 0.4%, 0.1% to 0.5%, 0.1% to 0.8%, 0.1% to 1%, 0.2% to 0.25%, 0.2% to 0.3%, 0.2% to 0.4%, 0.2% to 0.5%, 0.2% to 0.8%, 0.2% to 1%, or 0.2%) Treat the cells with X-100 at -4°C to 10°C (eg, 0°C to 4°C) for 0.5-10 min (eg, 1-5 min or 3 min) to permeabilize the cells.

[0211] In certain embodiments, cells are fixed and permeabilized by treating the cells with 80% methanol at -20°C for 10 minutes.

[0212] In certain embodiments, the cell is a naturally occurring cell or a recombinant cell, or a mixture of both.

[0213] In certain embodiments, the recombinant cell refers to a cell comprising a modified (e.g., artificially modified) nucleic acid molecule (e.g., gene) and / or its product (e.g., protein, RNA), wherein the modification includes but is not limited to increasing or decreasing the copy number of the endogenous gene of the cell, mutating the endogenous gene of the cell, upregulating, downregulating or silencing the expression of the endogenous gene product of the cell, introducing an exogenous nucleic acid molecule into the cell (the exogenous nucleic acid molecule is integrated into the genome of the cell or exists in a non-integrated form), etc.

[0214] In an eighth aspect, the present application provides a method for fixing and permeabilizing a cell nucleus, comprising the following steps:

[0215] (i) fixing the cell nucleus, wherein the fixing is selected from the group consisting of:

[0216] (a) fixing the cell nuclei by treating the cell nuclei with formaldehyde at a concentration of 0.05%-4% (e.g., 0.5%-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, or 1%) at 0°C to 30°C (e.g., 15°C to 28°C or 25°C) for 2-20 minutes (e.g., 5-15 minutes or 10 minutes); or,

[0217] (b) fixing the cell nuclei by treating the cell nuclei with paraformaldehyde at a concentration of 0.05% to 4% (e.g., 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, or 1.6%) at 0° C. to 30° C. (e.g., 15° C. to 28° C. or 25° C.) for 1 to 15 minutes (e.g., 1 to 10 minutes or 5 minutes);

[0218] as well as,

[0219] (ii) permeabilizing the cell nucleus by treating the cell nucleus with a permeabilization solution containing digitonin at -4°C to 10°C (e.g., 0°C to 4°C) for 0.5-10 min (e.g., 1-5 min or 3 min);

[0220] In certain embodiments, the permeabilization solution further comprises IGEPAL (e.g., CA-630) and / or Tween-20.

[0221] In certain embodiments, the concentration of digitonin in the permeabilization solution is 0.0005%-0.05% (eg, 0.0008%-0.005%, 0.0005%-0.002%, 0.0008%-0.002%, or 0.001%).

[0222] In certain embodiments, the permeabilization solution contains IGEPAL (e.g., CA-630) at a concentration of 0.005%-0.1% (e.g., 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0223] In certain embodiments, the concentration of Tween-20 in the permeabilization solution is 0.005%-0.1% (eg, 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0224] In certain embodiments, the cell nuclei are fixed by treating the cell nuclei with 1% formaldehyde for 10 min at room temperature, and after fixation, the cell nuclei are fixed with a solution containing 0.001% digitonin, 0.01% IGEPAL (e.g., The cell nuclei are permeabilized by treating the cell nuclei with a permeabilization solution containing CA-630) and 0.01% Tween-20 at 0° C. to 4° C. for 2-4 min (e.g., 3 min).

[0225] In certain embodiments, the cell nucleus is a cell nucleus derived from a naturally occurring cell or a cell nucleus derived from a recombinant cell, or a mixture of both.

[0226] In certain embodiments, the recombinant cell refers to a cell comprising a modified (e.g., artificially modified) nucleic acid molecule (e.g., gene) and / or its product (e.g., protein, RNA), wherein the modification includes but is not limited to increasing or decreasing the copy number of the endogenous gene of the cell, mutating the endogenous gene of the cell, upregulating, downregulating or silencing the expression of the endogenous gene product of the cell, introducing an exogenous nucleic acid molecule into the cell (the exogenous nucleic acid molecule is integrated into the genome of the cell or exists in a non-integrated form), etc.

[0227] In a ninth aspect, the present application provides a device for labeling nucleic acid molecules from cells or cell nuclei and / or constructing a nucleic acid molecule library, the device comprising:

[0228] Memory; and

[0229] A processor coupled to the memory, the processor being configured to execute the method described in any one of the first aspects and / or the method described in any one of the second aspects based on instructions stored in the memory.

[0230] In the tenth aspect, the present application provides a computer-readable storage medium having a computer program stored thereon, characterized in that when the program is executed by a processor, it implements any method of the above-mentioned first aspect and / or any method of the above-mentioned second aspect.

[0231] In the eleventh aspect, the present application also provides the use of the method of any one of the above-mentioned first aspects or the reagent composition of the above-mentioned fifth aspect or the kit of the sixth aspect, the method of the seventh aspect or the eighth aspect, the device of the ninth aspect or the computer-readable storage medium of the tenth aspect for constructing a nucleic acid molecule library or for performing transcriptome sequencing; or, the use of any one of the above-mentioned methods of the second aspect for performing transcriptome sequencing.

[0232] In certain embodiments, the present application provides the following embodiments:

[0233] Embodiment 1. A method for labeling a nucleic acid molecule from a cell or a cell nucleus, comprising the steps of:

[0234] (1) providing a plurality of fixed and permeabilized cells or cell nuclei, wherein the cells or cell nuclei contain nucleic acid molecules to be labeled; and,

[0235] a plurality of beads coupled to a plurality of first oligonucleotide molecules, wherein the first oligonucleotide molecules contain a first tag sequence;

[0236] Furthermore, the plurality of first oligonucleotide molecules on the same bead have the same first tag sequence, and the first oligonucleotide molecules on different beads have different first tag sequences;

[0237] (2) randomly distributing the plurality of beads and the plurality of cells or cell nuclei into different first discrete partitions, releasing the first oligonucleotide molecule from the beads and contacting the first oligonucleotide molecule with the cells or cell nuclei within the first discrete partitions, thereby generating a first nucleic acid molecule derived from the nucleic acid molecule to be labeled within the cells or cell nuclei, wherein the first nucleic acid molecule contains the first tag sequence or its complementary sequence;

[0238] (3) mixing the cells or cell nuclei containing the first nucleic acid molecule from different first discrete partitions and redistributing them into different second discrete partitions;

[0239] (4) contacting a second oligonucleotide molecule containing a second tag sequence with the first nucleic acid molecule within the second discrete partition to generate a second nucleic acid molecule containing the first tag sequence or its complementary sequence and the second tag sequence or its complementary sequence;

[0240] wherein the second oligonucleotide molecules in the same second discrete partition have the same second tag sequence, and the second oligonucleotide molecules in different second discrete partitions have different second tag sequences;

[0241] The cells are naturally occurring cells or recombinant cells, or a mixture of the two; the cell nuclei are cell nuclei derived from naturally occurring cells or recombinant cells, or a mixture of the two.

[0242] Embodiment 2. The method of embodiment 1, wherein the cells are fixed and permeabilized using methanol, or formaldehyde or paraformaldehyde and Triton X-100.

[0243] Embodiment 3 The method of embodiment 1, wherein the method uses formaldehyde or paraformaldehyde and digitonin to fix and permeabilize the cell nucleus;

[0244] In certain embodiments, the method further comprises administering IGEPAL (e.g., The cell nuclei were permeabilized with CA-630) and / or Tween-20.

[0245] Embodiment 4. The method of any one of embodiments 1-3, comprising one or more selected from the following:

[0246] (1) In step (1), providing at least two cells or cell nuclei; and / or providing at least two beads;

[0247] (2) The first discrete partition is a discrete micropore or discrete microdroplet

[0248] (3) the beads are coupled to at least two of the first oligonucleotide molecules;

[0249] (4) the bead is capable of releasing the first oligonucleotide molecule spontaneously or upon exposure to one or more stimuli;

[0250] (5) The beads are gel beads;

[0251] (6) In step (3), the cells or cell nuclei are divided into at least two of the second discrete partitions, wherein each of the second discrete partitions contains at least one cell or cell nucleus;

[0252] (7) The second discrete partitions are discrete wells in a porous plate;

[0253] (8) After step (3) and before step (4), the method further includes the steps of lysing cells and / or purifying the first nucleic acid molecule.

[0254] Embodiment 5. The method of any one of Embodiments 1-4, wherein the nucleic acid molecule to be labeled is mRNA, and the first oligonucleotide molecule is the first oligonucleotide molecule a.

[0255] Embodiment 6. The method of embodiment 5, wherein step (2) comprises the following steps:

[0256] (i) (a) reverse transcribing the nucleic acid molecule to be labeled with the first oligonucleotide molecule a within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the nucleic acid molecule to be labeled, formed using the first oligonucleotide molecule a as a reverse transcription primer, and a 3'-terminal overhang; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A to the cDNA chain generated in (a) and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the primer A comprises, from the 5' end to the 3' end, a consensus sequence O and a complementary sequence to the 3'-terminal overhang;

[0257] or,

[0258] (ii) (a) reverse transcribing the nucleic acid molecule to be labeled using primer B within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the nucleic acid molecule to be labeled formed using primer B as a reverse transcription primer, and a 3'-terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; and, (b) annealing the first oligonucleotide molecule a with the cDNA chain generated in (a) within the first discrete partition, and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3'-terminal overhang.

[0259] In certain embodiments, in said step (ii), step (ii)(a) is performed before or after partitioning said cells or cell nuclei into said first discrete partitions.

[0260] In certain embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in certain embodiments, the unique molecular tag sequence is located at the 3' end of the common sequence R1 or a partial sequence thereof.

[0261] In certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T.

[0262] In certain embodiments, the 3'-terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length; in certain embodiments, the 3'-terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0263] Embodiment 7. The method of embodiment 5 or 6, wherein step (4) comprises the following steps:

[0264] In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer C as primers, and the generated extension product is the second nucleic acid molecule;

[0265] Wherein, the second oligonucleotide molecule comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof.

[0266] Embodiment 8. The method of any one of Embodiments 1-4, wherein the nucleic acid molecule to be labeled is genomic DNA, and the first oligonucleotide molecule is the first oligonucleotide molecule b.

[0267] Embodiment 9. The method of embodiment 8, wherein step (2) comprises the following steps:

[0268] (a) incubating the nucleic acid molecule to be labeled with a transposase complex I; wherein the transposase complex I contains a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a first transferred strand and a second transferred strand, the first transferred strand comprising a transposase recognition sequence and a consensus sequence R2 or a partial sequence thereof, and the second transferred strand comprising a transposase recognition sequence and a consensus sequence R1 or a partial sequence thereof; and the incubation is performed under conditions that allow the nucleic acid molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strands to be ligated to the ends of the nucleic acid fragments (e.g., the 5' ends of the nucleic acid fragments); thereby generating double-stranded nucleic acid fragments whose 5' ends respectively contain the consensus sequence R2 or a partial sequence thereof and the consensus sequence R1 or a partial sequence thereof; and,

[0269] (b) Within the first discrete partition, the first oligonucleotide molecule b is connected to the double-stranded nucleic acid fragment generated in (a) (for example, by using a nuclease), and an extension reaction is performed to generate an extension product, which is the first nucleic acid molecule; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof and the first tag sequence.

[0270] In certain embodiments, the first nucleic acid molecule comprises a sequence derived from a genomic DNA fragment in an open chromatin region in the cell or cell nucleus.

[0271] In certain embodiments, step (a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0272] In certain embodiments, the 5' end of the consensus sequence R1 of the transposase complex I or a partial sequence thereof is phosphorylated.

[0273] Embodiment 10. The method of embodiment 8 or 9, wherein step (4) comprises the following steps:

[0274] In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer D as primers, and the generated extension product is the second nucleic acid molecule;

[0275] The second oligonucleotide molecule comprises, from the 5' end to the 3' end: the consensus sequence P2 or a partial sequence thereof, the second tag sequence, the consensus sequence R2 or a partial sequence thereof; and the primer D comprises the consensus sequence P1 or a partial sequence thereof.

[0276] Embodiment 11. The method according to any one of embodiments 1 to 4, wherein the nucleic acid molecules to be labeled are mRNA and genomic DNA, and the mRNA and genomic DNA have the same cell origin;

[0277] Furthermore, the first oligonucleotide molecule includes a first oligonucleotide molecule a and a first oligonucleotide molecule b, and the second oligonucleotide molecule includes a second oligonucleotide molecule a and a second oligonucleotide molecule b;

[0278] The beads are coupled to a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b at the same time; and the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence.

[0279] Embodiment 12. The method of embodiment 11, wherein step (2) comprises the following steps:

[0280] (A)(i)(a) reverse transcribing the mRNA molecule to be labeled with the first oligonucleotide molecule a within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled, formed using the first oligonucleotide molecule a as a reverse transcription primer, and a 3'-terminal overhang; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A to the cDNA chain generated in (a) and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the primer A comprises, from the 5' end to the 3' end, a consensus sequence O and a complementary sequence to the 3'-terminal overhang;

[0281] or,

[0282] (ii) (a) reverse transcribing the mRNA molecule to be labeled with primer B within the first discrete partition to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled, formed using primer B as a reverse transcription primer, and a 3'-terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; and, (b) annealing the first oligonucleotide molecule a with the cDNA chain generated in (a) within the first discrete partition, and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3'-terminal overhang;

[0283] and,

[0284] (B)(a) incubating the DNA molecule to be labeled with a transposase complex I; wherein the transposase complex I is as defined in Embodiment 9; and the incubation is performed under conditions that allow the DNA molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strands to be ligated to the ends of the nucleic acid fragments (e.g., the 5' ends of the nucleic acid fragments); thereby generating double-stranded nucleic acid fragments whose 5' ends contain the consensus sequence R2 or a partial sequence thereof and the consensus sequence R1 or a partial sequence thereof, respectively; and,

[0285] (b) within the same first discrete partition as (A), ligating the first oligonucleotide molecule b to the double-stranded nucleic acid fragment generated in (a), and performing an extension reaction to generate an extension product, which is the first nucleic acid molecule b; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0286] Wherein, the step (A) and the step (B) can be performed in any order (for example, (A) first and then (B), (B) first and then (A), or simultaneously).

[0287] In certain embodiments, in step (A)(ii), step (A)(ii)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0288] In certain embodiments, step (B)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.

[0289] In certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T.

[0290] In certain embodiments, the first nucleic acid molecule b comprises a sequence derived from a genomic DNA fragment in an open chromatin region in the cell or cell nucleus.

[0291] In certain embodiments, the 5' end of the consensus sequence R1 or a partial sequence thereof in the transposase complex I is phosphorylated.

[0292] In certain embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in certain embodiments, the unique molecular tag sequence is located at the 3' end of the common sequence R1 or a partial sequence thereof.

[0293] In certain embodiments, the 3'-terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length; in certain embodiments, the 3'-terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0294] Embodiment 13. The method of embodiment 11 or 12, wherein step (4) comprises the following steps:

[0295] (a) amplifying the first nucleic acid molecule a in the second discrete partition using the second oligonucleotide molecule a and primer C as primers, whereby the generated extension product is the second nucleic acid molecule a;

[0296] Wherein, the second oligonucleotide molecule a comprises, from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof;

[0297] and

[0298] (b) amplifying the first nucleic acid molecule b within the same second discrete partition using the second oligonucleotide molecule b and primer D as primers, whereby the generated extension product is the second nucleic acid molecule b;

[0299] Wherein, the second oligonucleotide molecule b comprises, from the 5' end to the 3' end: the consensus sequence P2 or a partial sequence thereof, the second tag sequence, the consensus sequence R2 or a partial sequence thereof; the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0300] Wherein, the step (a) and the step (b) can be performed in any order (for example, (a) first and then (b), (b) first and then (a), or simultaneously).

[0301] Embodiment 14. A method for constructing a nucleic acid molecule library, comprising:

[0302] (1) generating a plurality of labeled second nucleic acid molecules according to the method of any one of embodiments 1-13, and,

[0303] (2) recovering and / or combining a plurality of said second nucleic acid molecules,

[0304] Thus, a nucleic acid molecule library is obtained.

[0305] In certain embodiments, in step (2), the second nucleic acid molecules generated in a plurality of the second discrete partitions are recovered and / or pooled.

[0306] Embodiment 15. The method of embodiment 14, comprising:

[0307] (a) generating a plurality of labeled second nucleic acid molecules according to the method of any one of embodiments 5-7,

[0308] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions; and

[0309] (c) randomly fragmenting the second nucleic acid molecule and adding a linker sequence;

[0310] Thus, the sequence of the nucleic acid molecule library is obtained.

[0311] Embodiment 16. The method of embodiment 15, wherein the cell is a T cell or a B cell.

[0312] In certain embodiments, the method further comprises, after step (a) and before step (c), a step of enriching the target nucleic acid molecule; the target nucleic acid molecule is a second nucleic acid molecule comprising: (i) a nucleotide sequence encoding a T cell receptor (TCR) or a B cell receptor (BCR) or a partial sequence thereof (e.g., a V(D)J sequence), and / or (ii) a complementary sequence to (i).

[0313] Embodiment 17. The method according to any one of embodiments 14 to 16, wherein in step (c), the second nucleic acid molecule is randomly fragmented by a transposase and a linker sequence is added to the 5' end thereof.

[0314] In certain embodiments, the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.

[0315] Embodiment 18. The method of any one of Embodiments 14-17, further comprising step (d):

[0316] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c).

[0317] In certain embodiments, step (d) comprises: amplifying the product of step (c) using primer E and primer F, wherein primer E comprises a consensus sequence P1 and an optional third tag sequence, and primer F comprises from 5' to 3': a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, a consensus sequence R2 or a partial sequence thereof.

[0318] Embodiment 19. The method of embodiment 14, comprising:

[0319] (a) generating a plurality of labeled second nucleic acid molecules according to the method of any one of embodiments 8-10, and,

[0320] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions;

[0321] Thus, the sequence of the nucleic acid molecule library is obtained.

[0322] Embodiment 20. The method of Embodiment 19, further comprising step (c):

[0323] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

[0324] In certain embodiments, step (c) comprises amplifying the product of step (b) using primer E' and primer F', wherein primer E' comprises a consensus sequence P1 and an optional third tag sequence, and primer F' comprises a consensus sequence P2 and an optional fourth tag sequence.

[0325] Embodiment 21. The method of embodiment 14, comprising:

[0326] (a) generating a plurality of labeled second nucleic acid molecules according to the method of any one of embodiments 11-13, comprising a plurality of second nucleic acid molecules a and a plurality of second nucleic acid molecules b, and,

[0327] (b) recovering and / or combining a plurality of said second nucleic acid molecules; in certain embodiments, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions;

[0328] Thus, the sequence of the nucleic acid molecule library is obtained.

[0329] Embodiment 22. The method of embodiment 21, wherein, after step (b), the method further comprises step (c): randomly breaking the second nucleic acid molecule a and adding a linker sequence.

[0330] In certain embodiments, in step (c), the second nucleic acid molecule a is randomly fragmented by a transposase and a linker sequence is added to the 5' end thereof.

[0331] In certain embodiments, the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.

[0332] Embodiment 23. The method of embodiment 22, wherein, before step (c), the method further comprises specifically enriching the second nucleic acid molecule a from the product of step (b).

[0333] In certain embodiments, the method specifically amplifies and enriches the second nucleic acid molecule a by using a primer G carrying a biotin label.

[0334] In certain embodiments, the primer G contains the consensus sequence O or a partial sequence thereof, or the primer G contains the consensus sequence T or a partial sequence thereof.

[0335] In certain embodiments, the amplification and enrichment further comprises using a primer H, wherein the primer H comprises a consensus sequence P1 or a partial sequence thereof.

[0336] Embodiment 24. The method of embodiment 22 or 23, wherein the method further comprises step (d):

[0337] a step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c).

[0338] In certain embodiments, step (c) comprises: amplifying the product of step (c) using primer E and primer F, wherein primer E comprises a consensus sequence P1 and an optional third tag sequence, and primer F comprises from 5' to 3': a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, and a consensus sequence R2.

[0339] Embodiment 25. The method of any one of Embodiments 21-24, further comprising step (d)':

[0340] A step of purifying and / or amplifying the second nucleic acid molecule b containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

[0341] In certain embodiments, step (d)' comprises: amplifying the product of step (b) using primer E' and primer F', wherein primer E' comprises a consensus sequence P1 and an optional third tag sequence, and primer F' comprises a consensus sequence P2 and an optional fourth tag sequence.

[0342] Embodiment 26. A method for performing omics sequencing on a cell or a cell nucleus, comprising:

[0343] Constructing a nucleic acid molecule library according to the method described in any one of embodiments 14-25; and,

[0344] The nucleic acid molecule library is sequenced.

[0345] In certain embodiments, before sequencing, at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, 2-5, 2-10, 2-20, 2-30, 2-40 or 2-50 nucleic acid molecule libraries are combined and then sequenced; wherein each nucleic acid molecule library each has multiple nucleic acid molecules (i.e., amplification products), and the multiple nucleic acid molecules in the same library have the same third tag sequence or the same fourth tag sequence; and nucleic acid molecules derived from different libraries have different third tag sequences or different fourth tag sequences from each other.

[0346] Embodiment 27. A nucleic acid molecule library constructed by the method described in any one of embodiments 14-25.

[0347] Embodiment 28. A reagent composition having the characteristics selected from I, II and III:

[0348] (I) The reagent composition comprises a second oligonucleotide molecule a, wherein the sequence of the second oligonucleotide molecule a comprises, from the 5′ end to the 3′ end, a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof;

[0349] Furthermore, the reagent composition further comprises one or more selected from the following:

[0350] (Ia) a plurality of beads coupled with a plurality of first oligonucleotide molecules a, wherein the first oligonucleotide molecules a contain a first tag sequence;

[0351] Furthermore, the plurality of first oligonucleotide molecules a on the same bead have the same first tag sequence, and the first oligonucleotide molecules a on different beads have different first tag sequences;

[0352] In certain embodiments, the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; or (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3' end overhang of the cDNA;

[0353] In some embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in some embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof;

[0354] (Ib) primer A or primer B, wherein the primer A comprises a consensus sequence O and a complementary sequence to the 3'-end overhang of the cDNA from the 5' end to the 3' end, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0355] (Ic) primer C, wherein the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0356] (Id) primer E and / or primer F, wherein the primer E comprises a consensus sequence P1 and an optional third tag sequence, and the primer F comprises, from 5' to 3', a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, a consensus sequence R2 or a partial sequence thereof;

[0357] (Ie) a transposase complex II, comprising a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break a double-stranded nucleic acid; wherein the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;

[0358] In certain embodiments, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides; in certain embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang);

[0359] (II) the reagent composition comprises a second oligonucleotide molecule b, wherein the sequence of the second oligonucleotide molecule b comprises, from the 5′ end to the 3′ end, a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof;

[0360] Furthermore, the reagent composition further comprises one or more selected from the following:

[0361] (II-a) a plurality of beads coupled with a plurality of first oligonucleotide molecules b, wherein the first oligonucleotide molecules b contain a first tag sequence;

[0362] Furthermore, the plurality of first oligonucleotide molecules b on the same bead have the same first tag sequence, and the first oligonucleotide molecules b on different beads have different first tag sequences;

[0363] In certain embodiments, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0364] (II-b) transposase complex I, wherein the transposase complex I is as defined in embodiment 9;

[0365] (II-c) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0366] (II-d) primer E' and / or primer F', wherein the primer E' comprises a consensus sequence P1 and an optional third tag sequence, and the primer F' comprises a consensus sequence P2 and an optional fourth tag sequence;

[0367] (III) The reagent composition comprises a second oligonucleotide molecule a and a second oligonucleotide molecule b; wherein the sequence of the second oligonucleotide molecule a comprises, from the 5' end to the 3' end, a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof; and the sequence of the second oligonucleotide molecule b comprises, from the 5' end to the 3' end, a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof;

[0368] Furthermore, the reagent composition further comprises one or more selected from the following:

[0369] (III-a) a plurality of beads to which a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b are simultaneously coupled, wherein the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence, the first oligonucleotide molecules a on different beads have different first tag sequences, and the first oligonucleotide molecules b on different beads have different first tag sequences;

[0370] In certain embodiments, the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; or, (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3' end overhang of the cDNA; and / or, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence;

[0371] In some embodiments, the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the plurality of first oligonucleotide molecules a coupled to the same bead have unique molecular tag sequences that are different from each other; in some embodiments, the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof;

[0372] (III-b) Primer A or primer B, wherein the primer A comprises a consensus sequence O and a complementary sequence to the 3'-end overhang of the cDNA from its 5' end to its 3' end, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from its 5' end to its 3' end; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0373] (III-c) transposase complex I, wherein the transposase complex I is as defined in embodiment 9;

[0374] (III-d) primer C, the primer C comprising the consensus sequence O or a partial sequence thereof, or the primer C comprising the consensus sequence T or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0375] (III-e) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof;

[0376] (III-f) primer E and / or primer F, wherein the primer E comprises the consensus sequence P1 and an optional third tag sequence, and the primer F comprises, from 5' to 3', the consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, and the consensus sequence R2 or a partial sequence thereof;

[0377] (III-g) primer E' and / or primer F', wherein the primer E' comprises a consensus sequence P1 and an optional third tag sequence, and the primer F' comprises a consensus sequence P2 and an optional fourth tag sequence;

[0378] (III-h) primer G and / or primer H, wherein primer G carries a biotin label and contains a consensus sequence O or a partial sequence thereof or a consensus sequence T or a partial sequence thereof, and primer H contains a consensus sequence P1 or a partial sequence thereof; in certain embodiments, the consensus sequence O is identical or partially identical to the consensus sequence T;

[0379] (III-h) transposase complex II, comprising a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;

[0380] In certain embodiments, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides; in certain embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0381] Embodiment 29. The reagent composition of embodiment 28, further comprising a reagent for fixing and / or permeabilizing cells or cell nuclei.

[0382] In certain embodiments, the reagent composition further comprises methanol, formaldehyde and / or paraformaldehyde.

[0383] In certain embodiments, the reagent composition further comprises Triton X-100, digitonin, IGEPAL (e.g., CA-630), and / or Tween-20.

[0384] In certain embodiments, the reagent composition further comprises: an RNase inhibitor, mineral oil, a buffer, dNTPs, one or more nucleic acid polymerases (e.g., DNA polymerases; e.g., DNA polymerases having strand displacement activity and / or high fidelity), reagents for recovering or purifying nucleic acids (e.g., magnetic beads), a well plate, or any combination thereof.

[0385] In certain embodiments, the reagent composition further comprises reagents for sequencing; for example, reagents for next-generation sequencing.

[0386] Embodiment 30. A kit comprising: a multi-reaction system comprising a plurality of oligonucleotide molecules, each of the oligonucleotide molecules comprising a specific tag sequence;

[0387] Furthermore, in the multi-reaction system, the oligonucleotide molecules in each reaction system have the same tag sequence, and the oligonucleotide molecules in different reaction systems have different tag sequences.

[0388] In certain embodiments, the oligonucleotide molecule further comprises a consensus sequence P1 or a partial sequence thereof, or the oligonucleotide molecule further comprises a consensus sequence P2 or a partial sequence thereof.

[0389] In certain embodiments, the multi-reaction system comprises at least 2 (e.g., at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 20, at least 24, at least 50, at least 96, at least 100, at least 200, at least 384, at least 400, 2-5, 2-10, 2-50, 2-80, 2-100, 2-500, 2-10 3 2-10 4 2-10 5 2-10 6 ) a multi-reaction system containing oligonucleotides;

[0390] The multi-reaction system is preferably a multi-well plate, and the oligonucleotides can be free or immobilized in the reaction system.

[0391] Embodiment 31. A method for fixing and permeabilizing cells, comprising the steps of:

[0392] (i) treating the cells with 60%-100% (e.g., 60%-80%, 70%-80%, 70%-85%, 70%-90%, 75%-80%, 75%-85%, 75%-90%, 75%-100%, 80%-85%, 80%-90%, 80%-100%, or 80%) methanol at -40°C to -10°C (e.g., -25°C to -15°C or -20°C) for 5-30 min (e.g., 8-20 min or 10 min) to fix and permeabilize the cells;

[0393] or,

[0394] (ii) (a) fixing the cells by treating the cells with formaldehyde or paraformaldehyde at a concentration of 0.05% to 5% (e.g., 0.5% to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, or 1%) at 0°C to 37°C (e.g., 15°C to 30°C or 25°C) for 5-30 minutes (e.g., 5-20 minutes or 10 minutes); and,

[0395] (b) using Triton at a concentration of 0.05% to 2% (e.g., 0.05% to 0.2%, 0.05% to 0.25%, 0.05% to 0.3%, 0.05% to 0.5%, 0.05% to 0.8%, 0.05% to 1%, 0.1% to 0.2%, 0.1% to 0.25%, 0.1% to 0.3%, 0.1% to 0.4%, 0.1% to 0.5%, 0.1% to 0.8%, 0.1% to 1%, 0.2% to 0.25%, 0.2% to 0.3%, 0.2% to 0.4%, 0.2% to 0.5%, 0.2% to 0.8%, 0.2% to 1%, or 0.2%) Treat the cells with X-100 at -4°C to 10°C (eg, 0°C to 4°C) for 0.5-10 min (eg, 1-5 min or 3 min) to permeabilize the cells.

[0396] In certain embodiments, the cell is a naturally occurring cell or a recombinant cell, or a mixture of both.

[0397] Embodiment 32. A method for fixing and permeabilizing a cell nucleus, comprising the steps of:

[0398] (i) fixing the cell nucleus, wherein the fixing is selected from the group consisting of:

[0399] (a) fixing the cell nuclei by treating the cell nuclei with formaldehyde at a concentration of 0.05%-4% (e.g., 0.5%-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, or 1%) at 0°C to 30°C (e.g., 15°C to 28°C or 25°C) for 2-20 minutes (e.g., 5-15 minutes or 10 minutes); or,

[0400] (b) fixing the cell nuclei by treating the cell nuclei with paraformaldehyde at a concentration of 0.05% to 4% (e.g., 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, or 1.6%) at 0° C. to 30° C. (e.g., 15° C. to 28° C. or 25° C.) for 1 to 15 minutes (e.g., 1 to 10 minutes or 5 minutes);

[0401] as well as,

[0402] (ii) permeabilizing the cell nuclei by treating the cell nuclei with a permeabilization solution containing digitonin at -4°C to 10°C (eg, 0°C to 4°C) for 0.5-10 min (eg, 1-5 min or 3 min).

[0403] In certain embodiments, the permeabilization solution further comprises IGEPAL (e.g., CA-630) and / or Tween-20.

[0404] In certain embodiments, the concentration of digitonin in the permeabilization solution is 0.0005%-0.05% (eg, 0.0008%-0.005%, 0.0005%-0.002%, 0.0008%-0.002%, or 0.001%).

[0405] In certain embodiments, the permeabilization solution contains IGEPAL (e.g., CA-630) at a concentration of 0.005%-0.1% (e.g., 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0406] In certain embodiments, the concentration of Tween-20 in the permeabilization solution is 0.005%-0.1% (eg, 0.005%-0.05%, 0.008%-0.05%, 0.005%-0.02%, 0.008%-0.02%, or 0.01%).

[0407] In certain embodiments, the cell nucleus is a cell nucleus derived from a naturally occurring cell or a cell nucleus derived from a recombinant cell, or a mixture of both.

[0408] Embodiment 33. A device for labeling nucleic acid molecules from cells or cell nuclei and / or constructing a nucleic acid molecule library, the device comprising:

[0409] Memory; and

[0410] A processor coupled to the memory, the processor being configured to execute the method described in any one of embodiments 1-13 and / or the method described in any one of embodiments 14-25 based on instructions stored in the memory.

[0411] Embodiment 34. A computer-readable storage medium having a computer program stored thereon, characterized in that when the program is executed by a processor, the method of any one of Embodiments 1-13 and / or the method of any one of Embodiments 14-25 is implemented.

[0412] Embodiment 35. Use of the method of any one of embodiments 1-13, the kit of embodiment 28 or 29, the reagent composition of embodiment 30, the method of embodiment 31 or 32, the device of embodiment 33, or the computer-readable storage medium of embodiment 34 for constructing a nucleic acid molecule library or for performing transcriptome sequencing; or, use of the method of any one of embodiments 14-25 for performing transcriptome sequencing.

[0413] Definition of terms

[0414] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0415] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0416] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0417] As used herein, the term "pseudo single cell" refers to a situation in which a micro-reaction system (e.g., a water-in-oil droplet or a micropore) contains two or more cells in a transcriptomic experiment analyzing a single cell. In the case of "pseudo single cells," two or more cells in the same micro-reaction system (e.g., the same droplet or micropore) will be labeled with the same cell-specific label. This results in the inability to perform a "one-to-one" identification of each cell in the micro-reaction system using only the cell-specific label introduced by the micro-reaction system. Accordingly, the sequencing data generated by the "pseudo single cell" micro-reaction system cannot be used to analyze the transcriptome information of a single cell because it contains sequencing results derived from two or more cells. Therefore, in traditional high-throughput single-cell transcriptome sequencing methods, it is necessary to filter or remove the sequencing data generated by the "pseudo single cell" micro-reaction system from the sequencing data ultimately generated; and, in order to avoid a large amount of waste in sequencing data, it is necessary to reduce or control the quantity or ratio of the "pseudo single cell" micro-reaction system as much as possible. As used herein, the term "pseudomonas rate" refers to the ratio of "pseudomonas" microreaction systems (number) to all microreaction systems (number) containing cells.

[0418] As used herein, "cell throughput" refers to the number of cells that can be simultaneously labeled in a single library construction reaction for a given single-cell library construction technology protocol.

[0419] As used herein, "sample throughput" refers to the number of samples that can be simultaneously labeled in a single library construction reaction for a given single-cell library construction technology protocol.

[0420] As used herein, the cells or cell nuclei that can be used in the methods of the present invention can be any cell or cell nucleus of interest, for example, cancer cells, stem cells, neural cells, fetal cells, and immune cells or cell nuclei involved in an immune response. The cells / cell nuclei can be a mixture of cells / cell nuclei of the same type, or a mixture of completely heterogeneous cells / cell nuclei of different types. The different cell / cell nucleus types can include cells / cell nuclei of different tissues of an individual or cells / cell nuclei of the same tissues of different individuals, or cells / cell nuclei of microorganisms of different genera, species, strains, variants, or any combination of any or all of the foregoing. For example, the different cell / cell nucleus types can include normal cells / cell nuclei and cancer cells / cell nuclei of an individual; various cell / cell nuclei types obtained from human subjects, such as various immune cells / cell nuclei; a variety of different bacterial species, strains, and / or variants from environmental, forensic, microbiome, or other samples; or any other various mixtures of cell / cell nucleus types.

[0421] As used herein, a "library of nucleic acid molecules" refers to a collection or population of labeled nucleic acid fragments generated from a target nucleic acid molecule, wherein the combination of labeled nucleic acid fragments in the collection or population exhibits a sequence that is qualitatively and / or quantitatively representative of the sequence of the target nucleic acid molecule from which the labeled nucleic acid fragment was generated.

[0422] As used herein, "discrete partitions" refer to mutually independent spatial units, such as droplets or wells, that contain a substance of interest. Generally speaking, each discrete partition can keep its own contents separate from the contents of other discrete partitions. In some embodiments, the discrete partitions may also contain other substances distributed according to different needs, such as dyes, emulsifiers, surfactants, stabilizers, polymers, aptamers, reducing agents, initiators, biotin labels, fluorophores, buffers, acidic solutions, alkaline solutions, light-sensitive enzymes, pH-sensitive enzymes, aqueous buffers, detergents, ionic detergents, non-ionic detergents, and the like.

[0423] As used herein, "cDNA," "cDNA chain," or "cDNA molecule" refers to "complementary DNA" synthesized by extension of a primer annealed to an RNA molecule of interest, catalyzed by an RNA-dependent DNA polymerase or reverse transcriptase, using at least a portion of an RNA molecule of interest as a template (this process is also referred to as "reverse transcription"). The synthesized cDNA molecule is "homologous," "complementary," "base-paired," or "complexed" with at least a portion of the template.

[0424] As used herein, "transposase" refers to an enzyme that is capable of forming a functional complex with a composition comprising a transposon end (e.g., a transposon, a transposon end, a transposon end composition) and catalyzing the insertion or transposition of the composition comprising a transposon end into a double-stranded nucleic acid molecule (e.g., a DNA double strand, an RNA / cDNA hybrid double strand) incubated with the enzyme in a transposition reaction (e.g., an in vitro transposition reaction). Non-limiting examples of transposases include Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products, and derivatives having transposition activity (e.g., having higher transposition activity) of the above transposases.

[0425] For a variety of reasons, a nucleic acid or polynucleotide of the invention (e.g., a first oligonucleotide molecule, a second oligonucleotide molecule, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, a transferred strand, a non-transferred strand in a transposase complex) can include one or more modified nucleic acid bases, sugar moieties, or internucleoside linkages. For example, some reasons for using nucleic acids or polynucleotides comprising modified bases, sugar moieties, or internucleoside linkages include, but are not limited to: (1) alteration of the Tm; (2) altering the susceptibility of the polynucleotide to one or more nucleases; (3) providing a moiety for attaching a label; (4) providing a label or label quencher; or (5) providing a moiety for attaching another molecule in solution or bound to a surface, such as biotin. For example, in some embodiments, a nucleic acid or polynucleotide of the invention (e.g., a first oligonucleotide molecule, a second oligonucleotide molecule, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, a transferred strand in a transposase complex, a non-transferred strand) can be synthesized such that the random portion comprises one or more conformationally restricted nucleic acid analogs, such as, but not limited to, one or more ribonucleic acid analogs in which a ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom; In some embodiments, the 3' end of the oligonucleotide can be subjected to dideoxy treatment to render the 3' end unable to be extended; in some embodiments, the 5' end of the nucleic acid or polynucleotide of the present invention (e.g., the first oligonucleotide molecule, the second oligonucleotide molecule, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, the transferred chain and the non-transferred chain in the transposase complex) can be phosphorylated to enable the 5' end to be ligated to the 3' end of another oligonucleotide under the action of a nucleic acid ligase. In the methods of the present invention, for example, the nucleic acid base in the single nucleotide at one or more positions in a polynucleotide or oligonucleotide (e.g., a first oligonucleotide molecule, a second oligonucleotide molecule, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, the transferred strand, the non-transferred strand in the transposase complex) may include guanine, adenine, uracil, thymine, or cytosine, or alternatively, one or more of the nucleic acid bases may include a modified base such as, but not limited to, xanthine, allyamino-uracil, allyamino-thymidine, hypoxanthine, 2-aminoadenine, 5-propynyluracil, 5-propynylcytosine, 4-thiouracil, 6-thioguanine, aza- and deaza-uracils, thymidine, cytosine, adenine, or guanine. Additionally, they may comprise nucleic acid bases derivatized with a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quenching moiety, or some other moiety.With respect to the nucleic acids or polynucleotides of the invention (e.g., the first oligonucleotide molecule, the second oligonucleotide molecule, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, the transferred strand, the non-transferred strand in the transposase complex), one or more of the sugar moieties may comprise 2'-deoxyribose, or alternatively, one or more of the sugar moieties may comprise some other sugar moiety, such as, but not limited to, ribose or 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose, which provide resistance to some nucleases, or 2'-amino-2'-deoxyribose or 2'-azido-2'-deoxyribose, which can be labeled by reaction with a visible, fluorescent, infrared fluorescent, or other detectable dye, or a chemical having an electrophilic, photoreactive, alkyne, or other reactive chemical moiety. The internucleoside linkages of the nucleic acids or polynucleotides of the invention can be phosphodiester linkages, or alternatively, one or more of the internucleoside linkages can comprise modified linkages such as, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.

[0426] In the method of the present application, the first tag sequence, the second tag sequence, the third tag sequence, the fourth tag sequence, the unique molecule tag sequence, and the tag sequence are not limited by their composition or length, as long as they can play an identification role. In certain embodiments, the first tag sequence has a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, 3-8, 3-15, 3-25 or 3-50 nucleotides. In certain embodiments, the second tag sequence has a length of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 3-8, 3-15, 3-25 or 3-50 nucleotides. For example, the length of the first tag sequence is 4-8 nucleotides. In certain embodiments, the third tag sequence has a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 3-8, 3-15, 3-25, or 3-50 nucleotides. In certain embodiments, the fourth tag sequence has a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 3-8, 3-15, 3-25, or 3-50 nucleotides. In certain embodiments, the unique molecular tag sequence has a length of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, 5-8, 5-15, 5-25, or 5-50 nucleotides.

[0427] Similarly, in the method of the present application, consensus sequence R1, consensus sequence R2, consensus sequence O, consensus sequence P1, consensus sequence P2, primer A, primer B, primer C, primer E, primer F, primer D, primer E', primer F', primer G, primer H, transferred strands, non-transferred strands in the transposase complex, etc. are not limited by their composition or length. Those skilled in the art can reasonably adjust the length and / or composition of the sequences for various reasons, which will not be elaborated here.

[0428] It is easy for those skilled in the art to understand that in order to make the method of the present application directly applicable to the most mainstream single-cell omics platform 10X Genomics platform, therefore, in certain embodiments, the consensus sequence R1 is identical to the Read1 sequence of the 10X Genomics platform or its partial sequence or its complementary sequence. In certain embodiments, the consensus sequence R2 is identical to the Read2 sequence of the 10X Genomics platform or its partial sequence or its complementary sequence. In certain embodiments, the consensus sequence O is identical to the TSO sequence of the 10X Genomics platform or its partial sequence or its complementary sequence. In certain embodiments, the consensus sequence P1 is identical to the P5 sequence of the 10X Genomics platform or its partial sequence or its complementary sequence. In certain embodiments, the consensus sequence P2 is identical to the P7 sequence of the 10X Genomics platform or its partial sequence or its complementary sequence.

[0429] As used herein, the term "bead" generally refers to a particle. Beads can be porous, non-porous, solid, semi-solid, semi-fluid, or fluid. Beads can be magnetic or non-magnetic. In some embodiments, beads can be soluble, rupturable, or degradable. In some cases, beads can be non-degradable. In some embodiments, beads can be gel beads. Gel beads can be hydrogel beads. Gel beads can be formed from molecular precursors, such as polymers or monomeric substances. Semi-solid beads can be liposomal beads.

[0430] Advantageous Effects of the Invention

[0431] The present invention can simultaneously meet the following five points:

[0432] 1. Significantly reducing the empty rate of the microreactor system can increase the cell throughput of the existing single-cell omics library construction system based on the microreactor system by 10-100 times, reaching 100,000-1 million cells per reaction;

[0433] 2. Low pseudo-single cell rate. Based on the micro-reaction barcode labeling of cells, additional cell barcodes are introduced. Therefore, even if multiple cells enter the same micro-reaction system, they can be distinguished, and sequencing data belonging to the traditional "pseudo-single cells" can also be used;

[0434] 3. Compatible with whole cell and nucleus-based library construction;

[0435] 4. This solution is applicable to a variety of single-cell omics library construction technologies based on existing mature micro-reaction (e.g., water-in-oil droplets, nanopore) cell barcoding platforms, including single-cell 3' RNA-seq, single-cell 5' RNA-seq, single-cell VDJ-seq, and single-cell RNA and ATAC-seq multi-omics technologies. It is also applicable to other technology variants proposed based on the 10X Genomics platform for modality expansion and quality improvement, such as single-cell CUT&Tag.

[0436] 5. The data quality obtained by this protocol is close to that obtained by standard operation of the commercial 10x Genomics platform. Metrics include but are not limited to: number of genes detected, VDJ capture rate, and detected ATAC-seq signal.

[0437] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0438] Figure 1: Schematic diagram and library structure of the existing 10X Genomics single-cell 3' RNA-Seq library construction process. Specifically, a single-cell suspension, reverse transcription reaction solution, and 10X Genomics cell barcode-labeled microbeads are prepared on the 10X Genomics platform into oil-in-water droplets, each containing one cell and one magnetic bead. After the droplets are collected and reverse transcription and template displacement reactions are performed on a PCR instrument, the cell barcodes on the microbeads are loaded into the cell's cDNA product. Subsequent cDNA amplification, enzyme digestion, linker addition, and PCR amplification yield the final library. P5 and P7 at the two ends of the library are the sequencing adapter sequences of Illumina; the sample index on the right is the label sequence of the library, which is used to distinguish the sample source of the sequencing data; Read1 and Read2 at both ends of the library are the two primers for sequencing at both ends; the 10X Barcode on the left end of the library is the cell barcode, which can distinguish different single cells; UMI (unique molecular identifiers) single molecule barcodes are used to mark different mRNAs in the same cell; Poly (dT) is a polythymidine oligonucleotide, introduced during the mRNA reverse transcription process; the thin line in the middle is the transcriptome sequence.

[0439] Figure 2: Schematic diagram of an exemplary process principle of the present invention. Specifically, the present invention is based on a combination of cellular barcoding and post-combination labeling based on micro-reactions (e.g., the water-in-oil microdroplets shown in Figure 2). After completing the first round of barcoding in situ, the cells are re-mixed and redistributed, and a second round of labeling (96-384 types) is performed on the cells' nucleic acid molecules, enabling multiple novel single-cell omics library construction schemes.

[0440] Figure 3: Schematic diagram of exemplary single-cell transcriptome, VDJ library construction and library structure of the present invention. Specifically, the present invention prepares oil-in-water microdroplets of complete fixed cells / cell nuclei and 10X GENOMICS RNA microbeads with cell labels, and adds the first round of labels to the mRNA to be tested in situ in the cell through reverse transcription reaction (3'RNA) / template displacement reaction (5'RNA). The cells that have completed the first round of label loading are then released from the microdroplets, mixed thoroughly and divided into 96 / 384-well plates, and different sequencing primers with specific label sequences are added to each well. PCR amplification is performed so that cells in different wells are added with different second-round labels. Then, the amplified products are collected together for purification and further library construction, and finally the corresponding single-cell transcriptome library is obtained. Among them, the amplified products of the 5' end RNA of the single cell can be further enriched with VDJ to obtain a VDJ library. The final library structure is as follows: P5 and P7 at the two ends are Illumina sequencing adapter sequences; i7 on the right is the library index sequence, which is used to distinguish different sequencing libraries; Read1 and Read2 at both ends of the library are the two primers for sequencing at both ends; the 10X Barcode on the left end of the library is the cell barcode, which is the first round of cell labeling; the part marked with round 2 is the second round of cell labeling introduced by index PCR; UMI (unique molecular identifiers) single molecule barcodes are used to label different mRNAs in the same cell; TSO is the template substitution sequence; the unmarked part in the middle is the transcriptome sequence.

[0441] Figure 4: Schematic diagram of an exemplary single-cell multi-omics library construction and library structure of the present invention. Specifically, the present invention prepares oil-in-water microdroplets for complete fixed cells / cell nuclei and 10X GENOMICS RNA microbeads with cell labels, and adds the first round of labels to the mRNA to be tested by reverse transcription reaction (3'RNA) / gDNA in the open region of chromatin by linking reaction in situ in the cell. The cells that have completed the first round of label loading are then released from the microdroplets, mixed thoroughly and divided into 96 / 384-well plates, and different sequencing primers with specific label sequences are added to each well. PCR amplification is performed so that cells in different wells are added with different second-round labels. Next, the amplified products are collected together for purification and further library construction. Finally, the two products are enriched by the corresponding primers for cDNA and gDNA, respectively, and then further library construction is performed to obtain the corresponding single-cell transcriptome sequencing library and ATAC-seq sequencing library.

[0442] Figure 5: Sequencing results of mixed samples of human and mouse cell lines using the single-cell transcriptome method of the present invention.

[0443] FIG6 shows the results of sequencing human peripheral blood mononuclear cell samples fixed under different conditions using the transcriptome method of the present invention.

[0444] FIG7 shows the results of sequencing frozen human peripheral blood mononuclear cell samples using the single-cell 5' RNA-seq method of the present invention.

[0445] FIG8 : The results of sequencing human peripheral blood mononuclear cell samples using the single-cell VDJ-seq method of the present invention.

[0446] Figure 9: Sequencing results of frozen human kidney samples using the single-cell transcriptome + ATAC multi-omics method of the present invention.

[0447] Sequence information

[0448] A description of the sequences involved in this application is provided in the table below.

[0449] Table 1: Sequence information Note: “-s-” ​​indicates thiolation modification; N is independently selected from A, T, C or G; “5Phos” indicates phosphorylation modification; “3ddC” indicates 2',3'-dideoxycytidine-5'-monophosphate; N is independently selected from A, T, C and G.

[0450] In existing microreaction cell barcoding platforms, taking the 10X Genomics Chromium platform as an example, not all microreaction systems (GEMs, water-in-oil droplets) contain single cells as expected. Often, they contain two or more cells, a situation also known as "pseudomonocytes." In the case of "pseudomonocytes," two or more cells in the same GEM will be labeled with the same barcode. This results in the inability to perform a "one-to-one" identification of two or more cells in the GEM using only the barcode in the GEM. Accordingly, the sequencing data generated by "pseudomonocyte" GEMs cannot be used to analyze the transcriptome information of a single cell because it contains sequencing results from two or more cells. Therefore, the sequencing data generated by "pseudomonocyte" GEMs needs to be filtered or removed from the final sequencing data; and to avoid a large amount of sequencing data waste, the number or ratio of "pseudomonocyte" GEMs needs to be reduced or controlled as much as possible, which greatly limits the library construction throughput.

[0451] Based on the barcode labeling of cells in micro-reactions (e.g., water-in-oil droplets, nanopores), the present invention removes the cells / nuclei that have completed the first round of cell barcode loading in situ in the cell / nucleus in the micro-reaction system, mixes them thoroughly, and then divides them into several equal parts. Then, a second round of labeling is introduced on the nucleic acid molecules of the cell / nucleus through index PCR in the micro-system, and finally, two rounds of label information are used to jointly define a cell. Thus, the nucleic acid molecule library constructed using the method of the present invention has two rounds of cell labels, which makes it possible to split the sequencing data generated by "pseudo-single cells" and then accurately track and determine the cell source of the sequencing data. For example, although two or more cells in a "pseudo-single cell" micro-reaction system contain the same first round of cell barcode labels, the two or more cells each contain different second round of cell barcode labels. Thus, the sequencing data generated by each cell therein can be distinguished according to the second round of cell barcode labels, so that even sequencing data generated by "pseudo-single cells" can be used. This greatly reduces the negative impact of "pseudo-monocytes" that appear during the library construction process; similarly, it also greatly reduces the empty rate of the micro-reaction system during the library construction process, significantly reducing reagent waste and lowering costs.

[0452] In addition, the applicant also wishes to emphasize that, although the methods based on pre-labeling and microfluidic droplet high-throughput library construction technology can improve throughput and reduce the empty rate and pseudo-single cell rate of micro-reaction systems compared to existing traditional microfluidic droplet high-throughput library construction technology, such methods cannot be simultaneously applied to or improve multiple existing single-cell omics library construction technologies (single-cell 3' end RNA-seq, single-cell 5' end RNA-seq, single-cell VDJ-seq, single-cell RNA and ATAC-seq multi-omics technology, etc.), and such methods are complex to operate and have many steps, and the data quality is inferior to standard microfluidic droplet high-throughput library construction technology.

[0453] Therefore, existing high-throughput single-cell omics library construction technologies cannot simultaneously achieve the following: high data quality, high cell throughput, ease of operation, low cost, and low false single cell rate. Moreover, there is currently no method that can simultaneously apply or improve multiple existing single-cell omics library construction technologies. DETAILED DESCRIPTION

[0454] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0455] Unless otherwise specified, the molecular biology experimental methods used in the present invention are carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0456] The reagent information involved in the examples of this application is as follows:

[0457] Note: The reagents shown in the table are all commercially available.

[0458] Unless otherwise noted, the reagents used in the examples of this application have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise noted or clearly contradicted by the context, the reagents used in the examples of this application can be purchased commercially or prepared according to widely used formulas in the corresponding fields.

[0459] Example 1: Fixation and permeabilization of single cell suspension

[0460] Depending on the experimental needs, intact single cells from fresh tissues, fresh cell lines, fresh blood samples, primary cells, or frozen cell samples can be used to build libraries. Prior to library construction, the cells need to be fixed and permeabilized. This example used HeLa cell lines, NIH3T3 cell lines (purchased from the Chinese Academy of Sciences Cell Bank), and peripheral blood mononuclear cells (PBMCs) for the experiment.

[0461] The fixation and permeabilization steps are as follows:

[0462] Method 1: Fix and permeabilize with 80% methanol at -20℃ for 10 min.

[0463] Method 2: Fix with 1% formaldehyde at room temperature for 10 minutes, then centrifuge and remove the supernatant. Resuspend the cells in 0.2% Triton X-100 and permeabilize on ice for 3 minutes.

[0464] Method 3: Fix with 1% paraformaldehyde at room temperature for 10 minutes, then centrifuge and remove the supernatant. Resuspend the cells in 0.2% Triton X-100 and permeabilize on ice for 3 minutes.

[0465] After fixation, centrifuge at 500 g for 5 min at 4°C and remove the supernatant.

[0466] Example 2: Fixation and permeabilization of cell nuclei

[0467] Depending on experimental needs, cell nuclei from fresh tissues, frozen tissues, cell lines, blood samples, primary cells, and frozen cell samples can be used to construct libraries (cell nuclei can be extracted using conventional procedures widely used in the art). Prior to library construction, the cell nuclei need to be fixed and permeabilized.

[0468] The fixation and permeabilization steps are as follows:

[0469] Method 1: Fix with 1% formaldehyde at room temperature for 10 minutes.

[0470] Method 2: Fix with 1.6% paraformaldehyde at room temperature for 5 minutes.

[0471] After fixation, centrifuge at 600g at 4°C and discard the supernatant. Resuspend the cells in permeabilization buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL CA-630, 1% BSA, 0.001% digitonin, 1% SUPERase-In RNase Inhibitor), permeabilize on ice for 3 minutes, and then centrifuge and discard the supernatant.

[0472] Example 3: Preparation of single-end (i7-end) TN5 transposase complex

[0473] The single-end (i7-end) TN5 transposase complex will be used in the single-cell transcriptome library construction process of the present invention.

[0474] 1. Transposon preparation: Tn5-top_ME nucleotide (SEQ ID NO: 9) and Tn5-bottom_Read2N nucleotide (SEQ ID NO: 10) were prepared using TruePrep Dissolve the annealing buffer in the Tagment Enzyme kit to 100 μl, then mix the two nucleotides in a 1:1 volume ratio. For this example, take 10 μl of each nucleotide and mix thoroughly. Place the mixture in a PCR instrument and perform the following annealing reaction: 75°C for 15 minutes, 60°C for 10 minutes, 50°C for 10 minutes, 40°C for 10 minutes, and 25°C for 30 minutes. The annealed adapter mixture is the transposon and should be stored at -20°C.

[0475] 2. TN5 transposase complex embedding: using TruePrep Prepare the following reaction mixture using the TruePrep Tagment Enzyme (2 μg / μl) and Coupling Buffer from the Tagment Enzyme Kit: 10 μl of TruePrep Tagment Enzyme (2 μg / μl), 33 μl of Coupling Buffer, and 7 μl of transposon (obtained in the previous step). Mix thoroughly and incubate in a PCR instrument at 30°C for 1 hour. After completion of the reaction, the single-ended (i7-terminal) TN5 transposase complex will be obtained and stored at -20°C.

[0476] Example 4: Preparation of single-cell transcriptome library (including single-cell 3'RNA-

[0477] seq, human peripheral blood mononuclear cell single cell 5'RNA-seq library preparation)

[0478] 1. Water-in-oil microdroplet preparation and reverse transcription reaction barcode loading (first round of cell labeling): This example uses the 10X Genomics Chromium platform 10x Single Cell 5' RNA-seq and 10x Single Cell 3' RNA-seq systems to prepare water-in-oil microdroplets, assigning each microdroplet a unique label. The microbeads used for water-in-oil preparation and cell barcoding can be substituted with other platforms.

[0479] After counting the fixed and permeabilized single cell nuclei and permeabilized cell samples in the above example, the reverse transcription reaction solution was immediately prepared. The 10x Single Cell 5'RNA-seq reaction system was: 18.8μl RT Reagent B, 7.3μl Poly-dT RT Primer, 1.9μl Reducing Agent B, 2μl RT Enzyme C, 38.7ul fixed and permeabilized cell / cell nucleus suspension; the 10x Single Cell 3'RNA-seq reaction system was: 18.8μl RT Reagent B, 2.4μl Template Switch Oligo, 2μl Reducing Agent B, 8.7μl RT Enzyme C, 43.2ul fixed and permeabilized cell / cell nucleus suspension. Following the 10X Chromium Single Cell Reagent Kits User Guide, load 70µl of cell reaction solution, 50µl of 10X Single Cell Gel Beads, and 45µl of mineral oil onto a 10X Chip K (for 10x Single Cell 5' RNA-seq) or 10X Chip G (for 10x Single Cell 3' RNA-seq) chip. Perform a water-in-oil preparation on a 10X Genomics Chromium instrument. After the preparation, collect the water-in-oil product into a 200µl PCR tube and quickly place it in a thermal cycler. Incubate at 53°C for 45 minutes, then store at 4°C.

[0480] 2. Breaking the oil-in-water microdroplets to release cells and redistribute cells: The oil-in-water product that has completed the first round of barcode cell label loading is broken into oil-in-water microdroplets, the cells are removed from the aqueous phase and mixed thoroughly, and then the cell / nucleus suspension is dispensed into a 96-well plate.

[0481] 3. Cell lysis and purification: Place the 96-well plate containing the cells in a PCR instrument and incubate at 85°C for 5 minutes. Purification is then performed.

[0482] 4. Index PCR amplification reaction (loading the second round of cell labeling): Add the cDNA amplification reaction solution to the above-mentioned purified product. The reaction solution to be added to each well of the 96-well plate includes: 20ul KAPA HiFi HotStart 2X ReadyMix, 2ul 10uM Partial TSO / IS primer (SEQ ID NO: 2), and 2ul 10uM Truseq-i5 end-specific second index primer (sequence shown in SEQ ID NO: 5. There are 96 types of primers used in this example, and one is added to each well. The index sequences contained in the 96 primers are respectively selected from the sequence shown in SEQ ID NO: 7). After thorough mixing, quickly place in a PCR instrument for amplification.

[0483] 5. Purification of Index PCR cDNA amplification products: Collect the products from the above 96-well plate into a new EP tube, and purify and elute with 0.6x SPRIselect magnetic beads.

[0484] 6. Sequencing Library Construction: This example provides a library construction method using single-end transposase transposition with i7-end sequencing primers followed by amplification. First, 100 ng of the product from the previous step was transposed and fragmented using single-end (i7-end) TN5 transposase (prepared in Example 3). The reaction system consisted of: 10 μl of 5X Reaction (vazyme #S601-01), 5 μl of single-end (i7-end) TN5 transposase, and 100 ng of the Index PCR cDNA amplification product. The reaction mixture was thoroughly mixed and incubated at 55°C in a PCR instrument for 15 minutes. Purification and elution were then performed using 0.8x SPRIselect magnetic beads. The purified product was then amplified for sequencing using the following reaction system: 50 μl NEBNext High-Fidelity 2x PCR Master Mix, 5 μl 10 μM P5-terminal primer (SEQ ID NO: 1), Nextare-i7-terminal second index primer (sequence structure shown in SEQ ID NO: 6; the actual primer used in this example contained an index sequence selected from the sequence shown in SEQ ID NO: 8), and 40 μl of the transposition purified product. After thorough mixing, the product was placed in a PCR instrument and the reaction conditions were: 72°C for 5 minutes, 98°C for 45 seconds, 8 cycles of [98°C for 20 seconds, 60°C for 30 seconds, 72°C for 1 minute], 72°C for 5 minutes, and storage at 4°C.

[0485] 7. Sequencing Library Purification and Fragment Screening: Purify the product from the previous step and screen the fragments using 0.6X and 0.2X SPRIselect magnetic beads. Finally, a sequencing library with fragments of approximately 300-600 bp was obtained.

[0486] 8. Sequencing: The constructed library was sequenced using NovaSeq 6000 (Illumina, San Diego, CA) with a read length of 150 bp and paired-end sequencing, with 50,000 reads per cell.

[0487] Example 5: Preparation of single cell VDJ library (taking human peripheral blood mononuclear cells as an example)

[0488] The single-cell VDJ library preparation method provided in this embodiment is based on the purification of the single-cell 5'RNA-seq Index PCR cDNA amplification product shown in Example 4 by immune cell T cells and B cells, that is, 10X genomics chromium platform 10x Single Cell 5'RNA-seq is used to complete step 1-step 5 of Example 4, and the obtained two-round labeled cDNA amplification product is subjected to nested PCR amplification of VDJ conserved region specific primers to enrich for VDJ sequences. Since the two rounds of labels loaded on the cDNA are both located at the P5 end of the sequence (near the VDJ end), the enriched product still carries the same two rounds of cell labels as the single-cell 5'RNA-seq transcriptome. Then, a sequencing library is further constructed on the basis of the enriched VDJ. In this embodiment, PBMC derived from human peripheral blood is used as an example, and the VDJ libraries of T cells and B cells therein are constructed respectively.

[0489] Specific methods:

[0490] 1. Nested PCR specific enrichment of VDJ sequences: After the 10x Single Cell 5'RNA-seq Index PCR cDNA amplification product of human peripheral blood-derived PBMCs was purified in Example 4, the cDNA amplification product was enriched in two rounds of PCR using two sets of specific primers. The first round of nested PCR reaction system was: 50ul KAPA HiFi HotStart 2X ReadyMix, 5ul 10uM P5 end primer, 5ul 10uM human T Cell / B Cell Outer primer (there are 2 primers for T Cell (SEQ ID NOs: 11-12), 7 primers for B Cell (SEQ ID NOs: 15-21), the corresponding primers were mixed 1:1 before amplifying TCR / BCR), 5ul cDNA amplification product, 35ul nuclease-free water, mixed thoroughly and quickly placed in a PCR instrument. The reaction conditions were as follows: 98°C for 45s, TCR amplification for 11 cycles [98°C for 20s, 62°C for 30s, 72°C for 5s]. 1 min], 72°C for 1 min, and then stored at 4°C. Subsequently, the PCR amplification products were purified and fragments were selected using 0.5X and 0.3X SPRIselect magnetic beads and eluted with 40.5 μl of EB buffer. A second round of nested PCR amplification was then performed. The PCR reaction system was as follows: 50ul KAPA HiFi HotStart 2X ReadyMix, 5ul 10uM P5 end primer, 5ul 10uM human T Cell / B Cell Inner primer (there are 2 primers for T Cell (SEQ ID NOs:13-14) and 7 primers for B Cell (SEQ ID NOs:22-28). The corresponding primers were mixed 1:1 before amplifying TCR / BCR). 40ul of the first-round nested PCR amplification product was thoroughly mixed and quickly placed in a PCR instrument. The reaction conditions were as follows: 98°C for 45s, 9 cycles of TCR amplification (9 cycles of BCR amplification) [98°C for 20s, 62°C for 30s, 72°C for 1min], 72°C for 1min, and storage at 4°C. Finally, the final PCR product was purified and fragment selected using 0.5X and 0.25X SPRIselect magnetic beads and eluted with 30.5 μl of EB buffer. A 1 μl aliquot of the eluted product was measured for concentration using a Qubit assay. The remaining sample can be stored at -80°C for up to 3 months.

[0491] 2. Construction of VDJ sequencing library: Similar to Example 4, this example provides a library construction method in which a single-end transposase is transposed into an i7-end sequencing primer and then amplified. 100 ng of VDJ enriched product is taken and transposed with a single-end (i7-end) TN5 transposase (prepared in Example 3). The reaction system is: 10ul 5X Reaction (vazyme#S601-01), 5ul single-end (i7-end) TN5 transposase, 100ng of the above-mentioned Index PCR cDNA amplification product, and the total reaction system is 50ul. The insufficient volume is supplemented with nuclease-free water. After the reaction solution is thoroughly mixed, it is placed in a PCR instrument and incubated at 55°C for 5 minutes. The product is purified with 0.8x SPRIselect magnetic beads.

[0492] Finally, the beads were eluted with 40.5 μl of EB. The purified product was then amplified for sequencing using the following reaction system: 50 μl of NEBNext High-Fidelity 2x PCR Master Mix, 5 μl of 10 μM P5-terminal primer, 5 μl of 10 μM P5-terminal primer and Nextare-i7-terminal second index primer (the sequence structure is shown in SEQ ID NO: 6; the actual primer used in this example contained a random index sequence selected from SEQ ID NO: 8), and 40 μl of the transposition-purified product. After thorough mixing, the product was placed in a PCR instrument and the reaction conditions were: 72°C for 5 min, 98°C for 45 s, 7 cycles of [98°C for 20 s, 60°C for 30 s, 72°C for 1 min], 72°C for 5 min, and storage at 4°C.

[0493] 3. Sequencing Library Purification and Fragment Screening: Purify the product from the previous step and screen the fragments using 0.6X and 0.2X SPRIselect magnetic beads. Finally, a sequencing library with fragments of approximately 300-600 bp was obtained.

[0494] 4. Sequencing: The constructed library was sequenced using NovaSeq 6000 (Illumina, San Diego, CA) with a read length of 150 bp and paired-end sequencing, with 10,000 reads per cell.

[0495] Example 6: Preparation of a single-cell mRNA + genomic DNA multi-omics library (taking human kidney single-cell samples as an example)

[0496] This example utilizes the 10X genomics Chromium platform Single Cell Multiome ATAC+RNA-seq system to first complete the preparation of water-in-oil microdroplets and the loading of the first round of cell label barcodes. This is followed by a second round of cell labeling using index PCs for the transcriptome and chromatin access regions of the cell / nucleus, ultimately completing the construction of a single-cell multi-omics library. The water-in-oil preparation and cell barcode labeling microbeads can be replaced by other platforms. Specific methods:

[0497] 1. In situ transposition reaction: Perform in situ transposition reactions on fixed and permeabilized single nuclei and permeabilized cell samples from the previous examples according to the Chromium Next GEM Single Cell Multiome ATAC+Gene Expression User Guide. The reaction system consists of: 7 μl ATAC Buffer B, 3 μl ATAC Enzyme B, and 5 μl of the fixed cell / nucleus suspension. After thorough mixing, place the sample in a PCR instrument and incubate at 37°C for 60 minutes, followed by storage at 4°C. After completion of the reaction, specific adapter sequences will be introduced into the open chromatin regions of the sample nuclei.

[0498] 2. Oil-in-water droplet preparation and barcode loading (first round of cell labeling): After completing the in situ transposition reaction, prepare the reverse transcription and ligation reaction solution: 49.5μl Barcoding Reagent Mix, 1.9μl Reducing Agent B, 1.1μl Template Switch Oligo, and 7.5μl Barcoding Enzyme Mix. Mix the reaction mixture thoroughly and add it to the product from the previous step. Load 70μl of the cell reaction solution, 50μl of 10X Single Cell Gel Beads, and 45μl of mineral oil onto a 10X Chip J (add 50% glycerol to the remaining wells as specified in the manufacturer's instructions). Perform the oil-in-water preparation on a 10X genomics chromium instrument. After completion of the preparation, collect the oil-in-water product and quickly place it in a PCR instrument. Reaction conditions are as follows: 37°C for 45 minutes, 25°C for 30 minutes, and then store at 4°C.

[0499] 3. Breaking the water-in-oil microdroplets to release cells and redistribute cells: perform the same operation as described in step 2 of Example 4.

[0500] 4. Cell Lysis and Purification: Add 1µl of Proteinase K to each well of the aliquoted product, mix thoroughly, centrifuge briefly, and incubate in a PCR instrument at 55°C for 5 minutes. Prepare Dynabeads Cleanup Mix according to the 10X Chromium Single Cell Reagent Kits User Guide. Add 16µl of Dynabeads Cleanup Mix to each well of a 96-well plate for purification. Elute with 16.5µl of Elution Solution I per well, and transfer the eluate to a new 96-well plate. Purify the product again using 1.8x SPRIselect magnetic beads and elute with 16.5µl of EB. Transfer the eluted product to a new 96-well plate.

[0501] 5. Index PCR amplification reaction (loading the second round of cell labeling): cDNA and gDNA amplification reaction solutions were added to the purified products. The reaction solutions to be added to each well of the 96-well plate include: 25ul NEBNext High-Fidelity 2x PCR Master Mix, 2ul 10uM P5 end primer (SEQ ID NO: 1), 2ul 10uM Nextare-i7 end second index primer (sequence as SEQ ID NO: 6, there are 96 types of primers used in this example, one is added to each well, used to label gDNA from the open chromatin region, and the tag sequences contained in the 96 primers are respectively selected from the sequence shown in SEQ ID NO: 8), 2ul 10uM Partial TSO / IS primer (SEQ ID NO: 2), 2ul 10uM Truseq-i5 end specific second index primer (sequence as SEQ ID NO: 6). NO:5. 96 primers were used in this example, one of which was added to each well to label transcriptome cDNA. The tag sequences contained in the 96 primers were selected from the sequence shown in SEQ ID NO:7. After thorough mixing, the cells were quickly placed in a PCR instrument. The reaction conditions were as follows: 72°C for 5 min, 98°C for 3 min, 7 cycles of [98°C for 20 s, 63°C for 30 s, 72°C for 1 min], 72°C for 1 min, and storage at 4°C.

[0502] 6. Purification of Index PCR Products: Collect the products from the 96-well plate into a new Eppendorf tube, purify with 1.6x SPRIselect magnetic beads, and elute with 400 μl of EB. Purify the eluted product again with 1.6x SPRIselect magnetic beads and finally elute with 160 μl of EB. A 1 μl aliquot of the eluted product is collected and the concentration is measured using a Qubit assay. The remaining sample can be stored at -80°C for up to 3 months.

[0503] 7. cDNA Enrichment: 40 μl of the Index PCR product was enriched using biotin-modified primers. Add 60 μl of a reaction solution containing: 50 μl KAPA HiFi HotStart 2X ReadyMix, 5 μl 10 μM P5-terminal primer (SEQ ID NO: 1), and 5 μl 10 μM Bio-Partial TSO / IS primer (SEQ ID NO: 3). Mix thoroughly and quickly place in a PCR instrument. Reaction conditions are as follows: 98°C for 30 seconds, 6 cycles of [98°C for 20 seconds, 54°C for 30 seconds, and 72°C for 20 seconds], 72°C for 1 minute, and then store at 4°C. Next, enrich the biotin-labeled cDNA product using MyOne Streptavidin C1 beads (purify according to the MyOne Streptavidin C1 beads instructions). The C1 beads adsorbed with the cDNA amplification product were then resuspended in fresh PCR reaction solution (containing: 50 μl KAPA HiFi HotStart 2X ReadyMix, 5 μl 10 μM P5-terminal primer (SEQ ID NO: 1), 5 μl 10 μM Partial TSO / IS primer (SEQ ID NO: 2), and 40 μl nuclease-free water). After thorough mixing, further amplification was performed using the following reaction conditions: 98°C for 30 seconds, four cycles of [98°C for 20 seconds, 54°C for 30 seconds, and 72°C for 20 seconds], 72°C for 1 minute, and storage at 4°C. After the PCR reaction, the tubes were placed on a magnetic stand for 5 minutes. The supernatant was aspirated and purified using 0.6x SPRIselect magnetic beads and eluted with EB. A 1 μl aliquot of the eluted product was analyzed for concentration using a Qubit assay. The remaining sample can be stored at -80°C for up to 3 months.

[0504] 8. Construction of cDNA sequencing library and sequencing: Same as steps 6, 7, and 8 of Example 4.

[0505] 9. ATAC-seq Sequencing Library Construction: Prepare 40 µL of the purified Index PCR amplification product from Step 6 of this protocol for ATAC-seq sequencing library construction. Add 50 µL of KAPA HiFi HotStart2X ReadyMix, 5 µL of 10 µM P5 primer (SEQ ID NO: 1), and 5 µL of 10 µM P7 primer (SEQ ID NO: 4) to the 40 µL Index PCR amplification product. Mix thoroughly and perform PCR amplification. Reaction conditions: 98°C for 45 seconds, 7-10 cycles (depending on the number of cells loaded) of [98°C for 20 seconds, 67°C for 30 seconds, 72°C for 20 seconds], 72°C for 1 minute, and store at 4°C.

[0506] 10. ATAC-seq library purification and fragment screening: 0.4X and 1X SPRIselect magnetic beads were used to purify and screen the product from the previous step. A sequencing library with fragment sizes of approximately 200-700 bp was obtained.

[0507] 11. ATAC-se library sequencing: The constructed library was sequenced using NovaSeq 6000 (Illumina, San Diego, CA) with a read length of 50 bp and paired-end sequencing, with 25,000 reads per cell.

[0508] The experimental results of the embodiments of the present application are shown in Figures 5-9.

[0509] Figure 5 shows the results of sequencing a mixed sample of human and mouse cell lines using the single-cell transcriptome method of the present invention. Specifically, it is a scatter plot of the number of UMIs mapped to the genomes of different species in single cells, where the UMI number represents the number of sequenced cDNA molecules. Each dot in the figure represents a cell (a total of 6446 dots). Light-colored dots represent cells containing almost exclusively mouse cDNA, dark-colored dots represent cells containing almost exclusively human cDNA, and black dots represent contaminated cells (i.e., pseudomonocytes).

[0510] Figure 6 shows the results of sequencing human peripheral blood mononuclear cell samples fixed under different conditions using the transcriptome method of the present invention. A represents the number of UMIs detected after peripheral blood mononuclear cells were fixed using three conditions (methanol, 1% formaldehyde, and 1% paraformaldehyde); B represents the number of genes detected after peripheral blood mononuclear cells were fixed using three conditions (methanol, 1% formaldehyde, and 1% paraformaldehyde); C represents the visualization of unsupervised clustering of all cells under the three fixation conditions; and D represents the distribution of cell clusters under the three fixation conditions.

[0511] Figure 7 shows the results of sequencing frozen human peripheral blood mononuclear cell samples using the single-cell 5' RNA-seq method of the present invention (single experiment cell throughput: 118,819), specifically the cell clustering visualization results of single-cell 5' RNA-seq of frozen human peripheral blood mononuclear cells, showing that all 27 major cell types in the blood were detected using this method.

[0512] Figure 8 shows the results of sequencing human peripheral blood mononuclear cell samples using the single-cell VDJ-seq method of the present invention. A and C are visualization results of cells with detected BCR / TCR clones, black dots represent cells with detected BCR / TCR clones, and light dots represent cells without detected BCR / TCR. Cells with detected BCR clones completely overlap with the positions annotated as B cells in the single-cell transcriptome data of Figure 7, and cells with detected TCR clones completely overlap with the positions annotated as T cells in the single-cell transcriptome data of Figure 7. B and D are the proportions of B cells and T cells with detectable BCR and TCR clones, respectively.

[0513] Figure 9 shows the results of sequencing a frozen human kidney sample using the single-cell transcriptome + ATAC multi-omics method of the present invention. Figure A shows the visualization of cell clustering from the single-cell transposon of a frozen human kidney sample, demonstrating that all 12 major renal cell types were detected using this method; Figure B shows the number of genes detected in a single cell of the single-cell transcriptome of a frozen human kidney sample; Figure C shows the visualization of cell clustering from the single-cell ATAC-seq portion of a frozen human kidney sample, demonstrating clustering of 18 cell clusters; and Figure D shows the ATAC-seq peak information obtained for various cell types in the single-cell ATAC-seq portion.

[0514] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for labeling a nucleic acid molecule from a cell or a cell nucleus, comprising the following steps: (1) providing a plurality of fixed and permeabilized cells or cell nuclei, wherein the cells or cell nuclei contain nucleic acid molecules to be labeled; and, A plurality of beads coupled to a plurality of first oligonucleotide molecules, wherein: The first oligonucleotide molecule contains a first tag sequence; Furthermore, the plurality of first oligonucleotide molecules on the same bead have the same first tag sequence, and the first oligonucleotide molecules on different beads have first tag sequences different from each other; (2) randomly allocating the plurality of beads and the plurality of cells or cell nuclei to different first discrete partitions, releasing the first oligonucleotide molecule from the beads and contacting the first oligonucleotide molecule with the cells or cell nuclei in the first discrete partitions, thereby generating a first nucleic acid molecule derived from the nucleic acid molecule to be labeled in the cells or cell nuclei, wherein the first nucleic acid molecule contains the first tag sequence or its complementary sequence; (3) mixing the cells or cell nuclei containing the first nucleic acid molecule originating from different first discrete partitions and redistributing them into different second discrete partitions; (4) contacting a second oligonucleotide molecule containing a second tag sequence with the first nucleic acid molecule within the second discrete partition to generate a second nucleic acid molecule containing the first tag sequence or its complementary sequence and the second tag sequence or its complementary sequence; wherein the second oligonucleotide molecules of the same second discrete partition have the same second tag sequence, and the second oligonucleotide molecules of different second discrete partitions have different second tag sequences; Wherein, the cell is a naturally occurring cell or a recombinant cell, or a mixture of the two; the cell nucleus is a cell nucleus derived from a naturally occurring cell or a recombinant cell, or a mixture of the two.

2. The method of claim 1, wherein The nucleic acid molecule to be labeled is mRNA, and the first oligonucleotide molecule is the first oligonucleotide molecule a; Preferably, the step (2) comprises the following steps: (i)(a) in the first discrete partition, reversely transcribe the nucleic acid molecule to be labeled with the first oligonucleotide molecule a to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the nucleic acid molecule to be labeled formed by using the first oligonucleotide molecule a as a reverse transcription primer, and a 3' terminal overhang; wherein the first oligonucleotide molecule a comprises from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A with the cDNA chain generated in (a), and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the primer A comprises from the 5' end to the 3' end a consensus sequence O and a complementary sequence to the 3' terminal overhang; or, (ii) (a) in the first discrete partition, reversely transcribe the nucleic acid molecule to be labeled using primer B to generate a cDNA chain, wherein the cDNA chain contains a sequence that is formed by using primer B as a reverse transcription primer and is identical to the nucleic acid molecule to be labeled. A complementary cDNA sequence, and a 3' terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly (T) sequence from the 5' end to the 3' end; and, (b) within the first discrete partition, the first oligonucleotide molecule a is annealed with the cDNA chain generated in (a), and an extension reaction is performed to generate an extension product, wherein the extension product is the first nucleic acid molecule; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence and a complementary sequence to the 3' terminal overhang.

3. The method of claim 2, wherein The step (4) comprises the following steps: In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer C as primers, and the generated extension product is the second nucleic acid molecule; Wherein, the second oligonucleotide molecule comprises from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof.

4. The method of claim 1, wherein The nucleic acid molecule to be labeled is genomic DNA, and the first oligonucleotide molecule is a first oligonucleotide molecule b; Preferably, the step (2) comprises the following steps: (a) incubating the nucleic acid molecule to be labeled with a transposase complex I; wherein the transposase complex I contains a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break a double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a first transferred strand and a second transferred strand, the first transferred strand comprises a transposase recognition sequence and a consensus sequence R2 or a partial sequence thereof, and the second transferred strand comprises a transposase recognition sequence and a consensus sequence R1 or a partial sequence thereof; and the incubation is performed under conditions that allow the nucleic acid molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strands are connected to the ends of the nucleic acid fragments (e.g., the 5' ends of the nucleic acid fragments); thereby generating double-stranded nucleic acid fragments whose 5' ends contain the consensus sequence R2 or a partial sequence thereof and the consensus sequence R1 or a partial sequence thereof, respectively; and, (b) In the first discrete partition, the first oligonucleotide molecule b is connected to the double-stranded nucleic acid fragment generated in (a) (for example, by using a nuclease), and an extension reaction is performed to generate an extension product, which is the first nucleic acid molecule; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence.

5. The method of claim 4, wherein The step (4) comprises the following steps: In the second discrete partition, the first nucleic acid molecule is amplified using the second oligonucleotide molecule and primer D as primers, and the generated extension product is the second nucleic acid molecule; The second oligonucleotide molecule comprises from the 5' end to the 3' end: a consensus sequence P2 or a partial sequence thereof, the second tag sequence, a consensus sequence R2 or a partial sequence thereof; and the primer D comprises a consensus sequence P1 or a partial sequence thereof.

6. The method of claim 1, wherein The nucleic acid molecules to be labeled are mRNA and genomic DNA, and the mRNA and genomic DNA have the same cell source; Furthermore, the first oligonucleotide molecule includes a first oligonucleotide molecule a and a first oligonucleotide molecule b, and the second oligonucleotide molecule includes a second oligonucleotide molecule a and a second oligonucleotide molecule b; Wherein, the bead is coupled with a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b at the same time; and the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence; Preferably, the step (2) comprises the following steps: (A)(i)(a) in the first discrete partition, reversely transcribe the mRNA molecule to be labeled with the first oligonucleotide molecule a to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled formed by using the first oligonucleotide molecule a as a reverse transcription primer, and a 3' terminal overhang; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; and, (b) annealing primer A with the cDNA chain generated in (a), and performing an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the primer A comprises, from the 5' end to the 3' end, a consensus sequence O and a complementary sequence to the 3' terminal overhang; or, (ii) (a) in the first discrete partition, reversely transcribe the mRNA molecule to be labeled with primer B to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the mRNA molecule to be labeled formed by using primer B as a reverse transcription primer, and a 3' terminal overhang; wherein the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; and, (b) in the first discrete partition, anneal the first oligonucleotide molecule a with the cDNA chain generated in (a), and perform an extension reaction to generate an extension product, wherein the extension product is the first nucleic acid molecule a; wherein the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence R1 or a partial sequence thereof, the first tag sequence and a complementary sequence to the 3' terminal overhang; and, (B)(a) incubating the DNA molecule to be labeled with a transposase complex I; wherein the transposase complex I is as defined in claim 4; and the incubation is performed under conditions that allow the DNA molecule to be broken into nucleic acid fragments by the transposase complex I and the transferred strand to be connected to the end of the nucleic acid fragment (e.g., the 5' end of the nucleic acid fragment); thereby generating a double-stranded nucleic acid fragment having a consensus sequence R2 or a partial sequence thereof and a consensus sequence R1 or a partial sequence thereof at the 5' end, respectively; and, (b) in the first discrete partition that is the same as (A), connecting the first oligonucleotide molecule b with the double-stranded nucleic acid fragment generated in (a), and performing an extension reaction to generate an extension product, which is the first nucleic acid molecule b; wherein the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence; Wherein, the step (A) and the step (B) may be performed in any order (for example, (A) first and then (B), (B) first and then (A), or simultaneously).

7. The method of claim 6, wherein The step (4) comprises the following steps: (a) amplifying the first nucleic acid molecule a in the second discrete partition using the second oligonucleotide molecule a and primer C as primers, and the generated extension product is the second nucleic acid molecule a; Wherein, the second oligonucleotide molecule a comprises from the 5' end to the 3' end: the consensus sequence P1 or a partial sequence thereof, the second tag sequence, the consensus sequence R1 or a partial sequence thereof; the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof; and (b) amplifying the first nucleic acid molecule b in the same second discrete partition using the second oligonucleotide molecule b and primer D as primers, and the generated extension product is the second nucleic acid molecule b; Wherein, the second oligonucleotide molecule b comprises from the 5' end to the 3' end: the consensus sequence P2 or a partial sequence thereof, the second tag sequence, the consensus sequence R2 or a partial sequence thereof; the primer D comprises the consensus sequence P1 or a partial sequence thereof; Wherein, the step (a) and the step (b) may be performed in any order (for example, (a) first and then (b), (b) first and then (a), or simultaneously).

8. A method for constructing a nucleic acid molecule library, comprising: (1) generating a plurality of labeled second nucleic acid molecules according to the method according to any one of claims 1 to 7, and, (2) recovering and / or combining a plurality of said second nucleic acid molecules, Thereby obtaining a nucleic acid molecule library; Preferably, in step (2), the second nucleic acid molecules generated in a plurality of the second discrete partitions are recovered and / or combined.

9. The method of claim 8, comprising: (a) generating a plurality of labeled second nucleic acid molecules according to the method of claim 2 or 3, (b) recovering and / or combining a plurality of said second nucleic acid molecules; preferably, recovering and / or combining a plurality of said second nucleic acid molecules generated in said second discrete partitions; and (c) randomly breaking the second nucleic acid molecule and adding a linker sequence; Thus obtaining the nucleic acid molecule library sequence; Preferably, in step (c), the second nucleic acid molecule is randomly interrupted by a transposase and a linker sequence is added to its 5' end; preferably, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof; Preferably, the method further comprises step (d): A step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c).

10. The method of claim 8, comprising: (a) generating a plurality of labeled second nucleic acid molecules according to the method of claim 4 or 5, and, (b) recovering and / or combining a plurality of said second nucleic acid molecules; Thus obtaining the nucleic acid molecule library sequence; Preferably, the method further comprises step (c): A step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

11. The method of claim 8, comprising: (a) generating a plurality of labeled second nucleic acid molecules according to the method of claim 6 or 7, comprising a plurality of second nucleic acid molecules a and a plurality of second nucleic acid molecules b, and, (b) recovering and / or combining a plurality of said second nucleic acid molecules; Thus obtaining the nucleic acid molecule library sequence; Preferably, the method further comprises, after step (b), step (c): randomly interrupting the second nucleic acid molecule a and adding a linker sequence; Preferably, in the step (c), the second nucleic acid molecule a is randomly interrupted by a transposase and a linker sequence is added to its 5' end; Preferably, the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.

12. The method of claim 11, wherein The method further comprises step (d): A step of purifying and / or amplifying the nucleic acid molecule containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (c); Preferably, step (c) comprises: using primer E and primer F to amplify the product of step (c), wherein primer E comprises a consensus sequence P1 and an optional third tag sequence, and primer F comprises from 5' to 3': a consensus sequence P2 or its complementary sequence, an optional fourth tag sequence, and a consensus sequence R2.

13. The method of claim 11 or 12, wherein The method further comprises the step (d)': A step of purifying and / or amplifying the second nucleic acid molecule b containing the first tag or its complementary sequence and the second tag or its complementary sequence in the product of step (b).

14. A reagent composition having the characteristics selected from I, II and III: (I) the reagent composition comprises a second oligonucleotide molecule a, wherein the sequence of the second oligonucleotide molecule a comprises, from the 5' end to the 3' end, a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof; Furthermore, the reagent composition further comprises one or more selected from the following: (Ia) a plurality of beads coupled with a plurality of first oligonucleotide molecules a, wherein: The first oligonucleotide molecule a contains a first tag sequence; Furthermore, the plurality of first oligonucleotide molecules a on the same bead have the same first tag sequence, and the first oligonucleotide molecules a on different beads have first tag sequences different from each other; Preferably, the first oligonucleotide molecule a comprises from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof; sequence, the first tag sequence, and a poly(T) sequence; or, (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence of a cDNA 3' end overhang; (Ib) primer A or primer B, wherein the primer A comprises a consensus sequence O and a complementary sequence of the cDNA 3' end overhang from the 5' end to the 3' end, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly(T) sequence from the 5' end to the 3' end; (Ic) primer C, wherein the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof; (Id) a transposase complex II, wherein the transposase complex II contains a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break a double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; preferably, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof; (II) the reagent composition comprises a second oligonucleotide molecule b, the sequence of which comprises, from the 5' end to the 3' end, a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof; Furthermore, the reagent composition further comprises one or more selected from the following: (II-a) a plurality of beads coupled with a plurality of first oligonucleotide molecules b, wherein the first oligonucleotide molecules b contain a first tag sequence; Furthermore, the plurality of first oligonucleotide molecules b on the same bead have the same first tag sequence, and the first oligonucleotide molecules b on different beads have first tag sequences different from each other; Preferably, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence; (II-b) transposase complex I, the transposase complex I as defined in claim 4; (II-c) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof; (III) The reagent composition comprises a second oligonucleotide molecule a and a second oligonucleotide molecule b; wherein the sequence of the second oligonucleotide molecule a comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof, a second tag sequence, and a consensus sequence R1 or a partial sequence thereof; the sequence of the second oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P2 or a partial sequence thereof, a second tag sequence, and a consensus sequence R2 or a partial sequence thereof; Furthermore, the reagent composition further comprises one or more selected from the following: (III-a) a plurality of beads to which a plurality of the first oligonucleotide molecules a and a plurality of the first oligonucleotide molecules b are simultaneously coupled, wherein the plurality of the first oligonucleotide molecules a and the plurality of the first oligonucleotide molecules b on the same bead have the same first tag sequence, the first oligonucleotide molecules a on different beads have first tag sequences different from each other, and the first oligonucleotide molecules b on different beads have first tag sequences different from each other; Preferably, the first oligonucleotide molecule a comprises, from the 5' end to the 3' end: (i) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a poly(T) sequence; or, (ii) a consensus sequence R1 or a partial sequence thereof, the first tag sequence, and a complementary sequence to the 3' end overhang of the cDNA; and / or, the first oligonucleotide molecule b comprises, from the 5' end to the 3' end: a consensus sequence P1 or a partial sequence thereof and the first tag sequence; (III-b) Primer A or primer B, wherein the primer A comprises a consensus sequence O and a cDNA 3' end from the 5' end to the 3' end. The primer B comprises a complementary sequence of the end overhang, and the primer B comprises a consensus sequence T or a partial sequence thereof and a poly (T) sequence from the 5' end to the 3' end; (III-c) transposase complex I, the transposase complex I being as defined in claim 4; (III-d) primer C, the primer C comprises the consensus sequence O or a partial sequence thereof, or the primer C comprises the consensus sequence T or a partial sequence thereof; (III-e) primer D, wherein the primer D comprises the consensus sequence P1 or a partial sequence thereof; (III-f) Primer E and / or primer F, wherein the primer E comprises a consensus sequence P1 and an optional third tag sequence, and the primer F comprises from 5' to 3': a consensus sequence P2 or a complementary sequence thereof, an optional fourth tag sequence, a consensus sequence R2 or a partial sequence thereof; (III-g) Transposase complex II, wherein the transposase complex II contains a transposase and a transposition sequence that the transposase can recognize and bind to, and can cut or break a double-stranded nucleic acid; and the transposition sequence comprises a transferred strand and a non-transferred strand; wherein the transferred strand comprises a linker sequence; preferably, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof.

15. A kit comprising: a multi-reaction system containing a plurality of oligonucleotide molecules, each of which contains a specific tag sequence; Furthermore, in the multi-reaction system, the oligonucleotide molecules in each reaction system have the same tag sequence, and the oligonucleotide molecules in different reaction systems have different tag sequences from each other; The oligonucleotide molecule further comprises a consensus sequence P1 or a partial sequence thereof, or the oligonucleotide molecule further comprises a consensus sequence P2 or a partial sequence thereof; The multi-reaction system comprises at least 2 (e.g., at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 20, at least 24, at least 50, at least 96, at least 100, at least 200, at least 384, at least 400, 2-5, 2-10, 2-50, 2-80, 2-100, 2-500, 2-10 3 pcs, 2-10 4 pcs, 2-10 5 pcs, 2-10 6 ) a multi-reaction system containing oligonucleotides; The multi-reaction system is preferably a multi-well plate, and the oligonucleotides can be free or fixed in the reaction system.

16. A device for labeling nucleic acid molecules from cells or cell nuclei and / or constructing a nucleic acid molecule library, the device comprising: Memory; and A processor coupled to the memory, wherein the processor is configured to execute the method of any one of claims 1 to 7 and / or the method of any one of claims 8 to 13 based on instructions stored in the memory.

17. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the method of any one of claims 1 to 7 and / or the method of any one of claims 8 to 13 is implemented.

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