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WO2025059808A1 - Procédé et kit de marquage à haut rendement de molécules d'acide nucléique cellulaire - Google Patents

Procédé et kit de marquage à haut rendement de molécules d'acide nucléique cellulaire Download PDF

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Publication number
WO2025059808A1
WO2025059808A1 PCT/CN2023/119479 CN2023119479W WO2025059808A1 WO 2025059808 A1 WO2025059808 A1 WO 2025059808A1 CN 2023119479 W CN2023119479 W CN 2023119479W WO 2025059808 A1 WO2025059808 A1 WO 2025059808A1
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Prior art keywords
sequence
nucleic acid
primer
oligonucleotide
molecule
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English (en)
Chinese (zh)
Inventor
蒋岚
李芸
黄正
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Beijing Institute Of Genomics Chinese Academy Of Sciences China National Center For Bioinformation
Beijing Institute of Genomics of CAS
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Beijing Institute Of Genomics Chinese Academy Of Sciences China National Center For Bioinformation
Beijing Institute of Genomics of CAS
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Application filed by Beijing Institute Of Genomics Chinese Academy Of Sciences China National Center For Bioinformation, Beijing Institute of Genomics of CAS filed Critical Beijing Institute Of Genomics Chinese Academy Of Sciences China National Center For Bioinformation
Priority to PCT/CN2023/119479 priority Critical patent/WO2025059808A1/fr
Priority to CN202380014812.9A priority patent/CN118647729A/zh
Publication of WO2025059808A1 publication Critical patent/WO2025059808A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • 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.
  • high-throughput single-cell transcriptome sequencing technology high-throughput single-cell transcriptome sequencing technology
  • chromatin accessibility Assay for Transposase-Accessible Chromatin
  • single-cell omics sequencing technology has greatly deepened human understanding of cell diversity and heterogeneity, and has played a revolutionary role in the development of multiple biological and biomedical research fields such as developmental biology, tumors and other diseases, assisted reproduction, immunology, neuroscience, and microbiology.
  • Existing single-cell sequencing mainly includes single-cell genome sequencing, transcriptome sequencing, methylation sequencing, chromatin accessibility sequencing, and single-cell multi-omics sequencing containing the above omics information. Its essence is to reveal the genome, transcriptome, methylation, chromatin open state and other omics changes of single cells by analyzing the sequence, copy number, modification status, and interaction of DNA and RNA in a single cell.
  • High-throughput single-cell library construction technology currently mainly includes high-throughput single-cell library construction technology for cell barcode labeling in microfluidic droplets or microplates.
  • all the 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 disadvantages: low cell throughput, high library construction cost, high empty rate of micro-reaction system, and high rate of pseudo-single cells.
  • the present application provides a method for labeling a nucleic acid molecule from a cell or a cell nucleus, comprising the following steps:
  • 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;
  • 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 cell nucleus derived from a recombinant cell, or a mixture of the two.
  • 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 endogenous genes in the cell, mutating endogenous genes in the cell, upregulating or downregulating or silencing the expression of endogenous gene products in the cell, introducing exogenous nucleic acid molecules into the cell (the exogenous nucleic acid molecules are integrated into the genome of the cell or exist in a non-integrated form), etc.
  • a modified nucleic acid molecule e.g., gene
  • its product e.g., protein, RNA
  • 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 (e.g., modified endogenous nucleic acid molecules (e.g., genes) of the cell or introduced exogenous nucleic acid molecules contained in the cell).
  • modified nucleic acid molecules in the cell/cell nucleus e.g., modified endogenous nucleic acid molecules (e.g., genes) of the cell or introduced exogenous nucleic acid molecules contained in the cell.
  • cells or cell nuclei containing the first nucleic acid molecule originating from at least 2 e.g., at least 10 , at least 10 , at least 10 , at least 10 , at least 10 , at least 10, at least 10 , 2-10, 2-10 , 2-10 , 2-10 , 2-10, 2-10 or 2-10 ) of the first discrete partitions are mixed and redistributed to different second discrete partitions.
  • 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.
  • the first tag sequence and the second tag sequence can be used together to identify the cell from which the sequencing data originates.
  • 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.
  • a single said first discrete partition contains one said bead.
  • the first discrete partitions each independently contain one or more cells or cell nuclei.
  • 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 cell nuclei.
  • 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 cell nuclei.
  • 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.
  • each of the second discrete partitions independently contains 0-10 7 (e.g., 0-10, 0-10 2 , 0-10 3 , 0-10 4 , 0-10 5 , 0-10 6 , 0-10 7 , 1-10, 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.
  • 0-10 7 e.g., 0-10, 0-10 2 , 0-10 3 , 0-10 4 , 0-10 5 , 0-10 6 , 0-10 7 , 1-10, 1-10 2 , 1-10 3 , 1-10 4 , 1-10 5 , 1-10 6 , or 1-10 7
  • step (2) the method randomly distributes the plurality of beads and the plurality of cells or cell nuclei to different first discrete partitions by a microdroplet microfluidics system or a microplate system.
  • the droplet microfluidic system is selected from but not limited to: the microfluidic oil-in-water system of the 10X GENOMICS platform, the microfluidic system of the Fluidigm C1 platform, and the microfluidic system of the Biorad ddSEQ system.
  • the microplate system is selected from but not limited to: the microplate system of the BD Rhapsody platform and the microplate system of the Neocell platform.
  • the method uses methanol to fix and permeabilize the cells, or uses formaldehyde or paraformaldehyde and Triton X-100 to fix and permeabilize the cells.
  • the method fixes and permeabilizes the cells by a treatment selected from the group consisting of:
  • the concentration of use is 0.05%-2% (for example, 0.05%-0.2%, 0.05%-0.25%, 0.05%-0.3%, 0.05%-0.5%, 0.05%-0.8%, 0.05%-1%, 0.1%-0.2%, 0.1%-0.25%, 0.1%-0.3%, 0.1%-0.4%,
  • the cells are permeabilized by treating the cells with Triton X-100 (e.g., 0.1%-0.5%, 0.1%-0.8%, 0.1%-1%, 0.2%-0.25%, 0.2%-0.3%, 0.2%-0.4%, 0.2%-0.5%, 0.2%-0.8%, 0.2%-1% or 0.2%) 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).
  • Triton X-100 e.g., 0.1%-0.5%, 0.1%-0.8%, 0.1%-1%, 0.2%-0.25%, 0.2%-0.3%, 0.2%-0.4%, 0.2%
  • cells are fixed and permeabilized by treating the cells with 80% methanol for 10 min at -20°C.
  • the methods use formaldehyde or paraformaldehyde and digitonin to fix and permeabilize the nuclei.
  • the method further comprises administering IGEPAL (e.g., CA-630) and/or Tween-20 to permeabilize the cell nuclei.
  • IGEPAL e.g., CA-630
  • Tween-20 e.g., Tween-20
  • the method fixes and permeabilizes the cell nuclei by a treatment selected from the group consisting of:
  • the permeabilization solution further comprises IGEPAL (e.g., CA-630) and/or Tween-20.
  • IGEPAL e.g., CA-630
  • Tween-20 e.g., Tween-20
  • 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%).
  • IGEPAL e.g., CA-630
  • 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%.
  • 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%).
  • 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 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).
  • the method of labeling nucleic acid molecules from cells or cell nuclei comprises one or more selected from the following:
  • step (1) at least 2 (e.g., at least 10, at least 102 , at least 103, at least 104 ) at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , 2-10, 2-10 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, 2-10 2 , 2-10 3 , 2-10 4 , 2-10 5 , 2-10 6 , 2-10 7 , 2-10 8 or 2-10 9 ) beads;
  • the first discrete partitions are discrete micropores or discrete microdroplets (e.g., water-in-oil droplets);
  • the beads are coupled to at least 2 (e.g., at least 10, at least 10, at least 10 , at least 10 , at least 10 , at least 10 , at least 10 , at least 10 , at least 10, 2-10 , 2-10 , 2-10, 2-10, 2-10, 2-10 , 2-10 or 2-10 ) of the first oligonucleotide molecules;
  • 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.);
  • one or more stimuli e.g., temperature change, pH change, exposure to a specific chemical or phase, exposure to light, a reducing agent, etc.
  • the beads are gel beads
  • 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 ) of the second discrete partitions, wherein each of the second discrete partitions contains at least one cell or cell nucleus;
  • the second discrete partitions are discrete holes in a porous plate
  • step (3) and before step (4) the method further includes the steps of lysing cells and/or purifying the first nucleic acid molecule.
  • the nucleic acid molecule to be labeled is mRNA
  • the first oligonucleotide molecule is the first oligonucleotide molecule a.
  • the step (2) comprises the following steps:
  • the primer B comprises a common sequence T or its (a) 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' end overhang.
  • an overhang can be formed or added at the 3' end of the cDNA chain by using a reverse transcriptase with terminal transfer activity.
  • step (ii)(a) is performed before or after assigning said cells or cell nuclei to said first discrete partitions.
  • 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 different from each other.
  • the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof.
  • the consensus sequence O is identical or partially identical to the consensus sequence T.
  • the 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.
  • the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).
  • step (4) comprises the following steps:
  • 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;
  • 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.
  • the nucleic acid molecule to be labeled is genomic DNA
  • the first oligonucleotide molecule is the first oligonucleotide molecule b.
  • the step (2) comprises the following steps:
  • 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).
  • the extension product 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.
  • step (a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.
  • step (4) comprises the following steps:
  • 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: 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.
  • the nucleic acid molecules to be labeled are mRNA and genomic DNA, and the mRNA and genomic DNA have the same cell source;
  • the first oligonucleotide molecule includes a first oligonucleotide molecule a and a first oligonucleotide molecule b
  • the second oligonucleotide molecule includes a second oligonucleotide molecule a and a second oligonucleotide molecule b;
  • 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.
  • 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
  • the first oligonucleotide molecule b in the first discrete partition that is the same as (A), the first oligonucleotide molecule b is connected to the double-stranded nucleic acid fragment generated in (a), and an extension reaction is performed to generate an extension product, wherein the extension product 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;
  • 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).
  • step (A)(ii)(a) is performed before or after assigning said cells or cell nuclei to said first discrete partitions.
  • step (B)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.
  • the consensus sequence O is identical or partially identical to the consensus sequence T.
  • 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.
  • the 5' end of the consensus sequence R1 or a partial sequence thereof in the transposase complex I is phosphorylated.
  • 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 different from each other.
  • the unique molecular tag sequence is located at the 3' end of the consensus sequence R1 or a partial sequence thereof.
  • the 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.
  • the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).
  • step (4) comprises the following steps:
  • 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;
  • 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;
  • 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).
  • library construction methods of the present invention are respectively:
  • the present application also provides a method for constructing a nucleic acid molecule library, which comprises:
  • nucleic acid molecule library is obtained.
  • step (2) the second nucleic acid molecules generated in a plurality of the second discrete partitions are recovered and/or combined.
  • the method comprises:
  • sequence of the nucleic acid molecule library is obtained.
  • the cell is a T cell or a B cell.
  • 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 of (i).
  • step (c) the second nucleic acid molecule is randomly fragmented by a transposase and an adapter sequence is added to its 5' end.
  • the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.
  • the method further comprises step (d):
  • 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.
  • the method comprises:
  • nucleic acid molecule to be labeled is genomic DNA " of the first aspect above, and,
  • sequence of the nucleic acid molecule library is obtained.
  • the method further comprises step (c):
  • 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.
  • the method comprises:
  • 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,
  • sequence of the nucleic acid molecule library is obtained.
  • the method further comprises, after step (b), step (c): randomly breaking the second nucleic acid molecule a and adding a linker sequence.
  • step (c) the second nucleic acid molecule a is randomly fragmented by a transposase and a linker sequence is added to its 5' end.
  • the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.
  • the method before step (c), further comprises specifically enriching the second nucleic acid molecule a from the product of step (b).
  • the method specifically amplifies and enriches the second nucleic acid molecule a by using a primer G carrying a biotin label.
  • 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.
  • the amplification and enrichment further comprises using a primer H, wherein the primer H comprises a consensus sequence P1 or a partial sequence thereof.
  • the method further comprises step (d):
  • 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.
  • the method further comprises step (d)':
  • 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.
  • the present application also provides a method for performing omics sequencing on a cell or a cell nucleus, comprising:
  • the nucleic acid molecule library is sequenced.
  • nucleic acid molecule libraries 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.
  • the present application also provides a nucleic acid molecule library, which is constructed by the method described in any one of the second aspects above.
  • the present application also provides a reagent composition having characteristics selected from I, II and III:
  • the reagent composition comprises a second oligonucleotide molecule a, the sequence of which 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 reagent composition further comprises one or more selected from the following:
  • 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;
  • 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;
  • 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 common sequence R1 or a partial sequence thereof;
  • 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
  • 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
  • the consensus sequence O is identical or partially identical to the consensus sequence T
  • 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;
  • 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;
  • transposase complex II 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; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;
  • 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 some embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., CCC overhang);
  • 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;
  • the reagent composition further comprises one or more selected from the following:
  • 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;
  • 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;
  • 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 described in the first aspect;
  • primer D comprises the consensus sequence P1 or a partial sequence thereof
  • primer E' comprises a consensus sequence P1 and an optional third tag sequence
  • primer F' comprises a consensus sequence P2 and an optional fourth tag sequence
  • 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;
  • the reagent composition further comprises one or more selected from the following:
  • 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;
  • 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 common sequence R1 or a partial sequence thereof;
  • 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
  • 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
  • the consensus sequence O is identical or partially identical to the consensus sequence T
  • 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 described in the first aspect;
  • 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;
  • primer D comprises the consensus sequence P1 or a partial sequence thereof
  • 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;
  • 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;
  • (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 comprises 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;
  • 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; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;
  • 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. CCC overhang).
  • the reagent composition further comprises a reagent for fixing and/or permeabilizing cells or cell nuclei.
  • the reagent composition further comprises methanol, formaldehyde and/or paraformaldehyde.
  • the reagent composition further comprises Triton X-100, digitonin, IGEPAL (e.g., CA-630), and/or Tween-20.
  • 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.
  • an RNase inhibitor e.g., 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
  • the reagent composition further comprises reagents for sequencing, such as reagents for next-generation sequencing.
  • the present application also provides a kit, which comprises: a multi-reaction system containing a plurality of oligonucleotide molecules, each of which contains a specific tag sequence;
  • the oligonucleotide molecules in each reaction system have the same tag sequence, and the oligonucleotide molecules in different reaction systems have different tag sequences.
  • 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 multiple 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 ) multiple reaction systems containing oligonucleotides;
  • the multi-reaction system is preferably a multi-well plate, and the oligonucleotides can be free or fixed in the reaction system.
  • the present application provides a method for fixing and permeabilizing cells, comprising the following steps:
  • cells are fixed and permeabilized by treating the cells with 80% methanol for 10 min at -20°C.
  • the cell is a naturally occurring cell or a recombinant cell, or a mixture of both.
  • 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 endogenous genes in the cell, mutating endogenous genes in the cell, upregulating or downregulating or silencing the expression of endogenous gene products in the cell, introducing exogenous nucleic acid molecules into the cell (the exogenous nucleic acid molecules are integrated into the genome of the cell or exist in a non-integrated form), etc.
  • a modified nucleic acid molecule e.g., gene
  • its product e.g., protein, RNA
  • the present application provides a method for fixing and permeabilizing a cell nucleus, comprising the following steps:
  • the permeabilization solution further comprises IGEPAL (e.g., CA-630) and/or Tween-20.
  • IGEPAL e.g., CA-630
  • Tween-20 e.g., Tween-20
  • 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%).
  • IGEPAL e.g., CA-630
  • 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%.
  • 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%).
  • 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 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).
  • 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.
  • 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 exogenous nucleic acid molecules into the cell (the exogenous nucleic acid molecules are integrated into the genome of the cell or exist in a non-integrated form), etc.
  • a modified nucleic acid molecule e.g., gene
  • its product e.g., protein, RNA
  • 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:
  • 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.
  • 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.
  • the present application also provides the use of any one of the methods of the first aspect above, the reagent composition of the fifth aspect above, the kit of the sixth aspect, the method of the seventh aspect, 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 methods of the second aspect above for performing transcriptome sequencing.
  • the present application provides the following embodiments:
  • Embodiment 1 A method for labeling a nucleic acid molecule from a cell or a cell nucleus, comprising the following steps:
  • 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;
  • the cell is a naturally occurring cell or a recombinant cell, or a mixture of the two; the cell nucleus is derived from The nucleus of a naturally occurring cell or a nucleus derived from a recombinant cell, or a mixture of both.
  • Implementation Option 2 The method of Implementation Option 1, wherein the method uses methanol to fix and permeabilize the cells, or uses formaldehyde or paraformaldehyde and Triton X-100 to fix and permeabilize the cells.
  • Embodiment 3 The method of embodiment 1, wherein the method uses formaldehyde or paraformaldehyde and digitonin to fix and permeabilize the cell nucleus;
  • the method further comprises administering IGEPAL (e.g., CA-630) and/or Tween-20 to permeabilize the cell nuclei.
  • IGEPAL e.g., CA-630
  • Tween-20 e.g., Tween-20
  • Embodiment 4 The method of any one of embodiments 1-3, comprising one or more selected from the following:
  • step (1) providing at least 2 cells or cell nuclei; and/or providing at least 2 beads;
  • the first discrete partition is a discrete micropore or a discrete microdroplet
  • the beads are coupled to at least two of the first oligonucleotide molecules
  • the bead is capable of releasing the first oligonucleotide molecule spontaneously or upon exposure to one or more stimuli;
  • the beads are gel beads
  • step (3) the cells or cell nuclei are distributed into at least two of the second discrete partitions, wherein each of the second discrete partitions contains at least one cell or cell nucleus;
  • the second discrete partitions are discrete holes in a porous plate
  • step (3) and before step (4) the method further includes the steps of lysing cells and/or purifying the first nucleic acid molecule.
  • 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.
  • Embodiment 6 The method of embodiment 5, wherein step (2) comprises the following steps:
  • the nucleic acid molecule to be labeled is reverse transcribed with primer B 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 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, 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
  • step (ii)(a) is performed before or after assigning said cells or cell nuclei to said first discrete partitions.
  • the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the multiple 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 common sequence R1 or a partial sequence thereof.
  • the consensus sequence O is identical or partially identical to the consensus sequence T.
  • the 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 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).
  • Embodiment 7 The method of embodiment 5 or 6, wherein step (4) comprises the following steps:
  • 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;
  • 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.
  • 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.
  • Embodiment 9 The method of embodiment 8, wherein step (2) comprises the following steps:
  • 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).
  • 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.
  • 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.
  • step (a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.
  • the 5' end of the consensus sequence R1 of the transposase complex I or a portion thereof is phosphorylated of.
  • Embodiment 10 The method of embodiment 8 or 9, wherein step (4) comprises the following steps:
  • 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: 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.
  • 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 source;
  • the first oligonucleotide molecule includes a first oligonucleotide molecule a and a first oligonucleotide molecule b
  • the second oligonucleotide molecule includes a second oligonucleotide molecule a and a second oligonucleotide molecule b;
  • 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.
  • Embodiment 12 The method of embodiment 11, wherein 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
  • transposase complex I (B) (a) incubating the DNA molecule to be labeled with 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 strand to be connected to the end of the nucleic acid fragment (e.g., the 5' end of the nucleic acid fragment); 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,
  • the first oligonucleotide molecule b is combined with the oligonucleotide molecule in (a) The generated double-stranded nucleic acid fragments are connected and extended 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;
  • 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).
  • step (A)(ii)(a) is performed before or after assigning said cells or cell nuclei to said first discrete partitions.
  • step (B)(a) is performed before or after partitioning the cells or cell nuclei into the first discrete partitions.
  • the consensus sequence O is identical or partially identical to the consensus sequence T.
  • 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.
  • the 5' end of the consensus sequence R1 or a partial sequence thereof in the transposase complex I is phosphorylated.
  • the first oligonucleotide molecule a further comprises a unique molecular tag sequence, and the multiple 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 common sequence R1 or a partial sequence thereof.
  • the 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 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).
  • Embodiment 13 The method of embodiment 11 or 12, wherein step (4) comprises the following steps:
  • 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;
  • 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;
  • 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).
  • Embodiment 14 A method for constructing a nucleic acid molecule library, comprising:
  • nucleic acid molecule library is obtained.
  • step (2) the second nucleic acid molecules generated in a plurality of the second discrete partitions are recovered and/or combined.
  • Embodiment 15 The method of embodiment 14, comprising:
  • sequence of the nucleic acid molecule library is obtained.
  • Embodiment 16 The method of Embodiment 15, wherein the cell is a T cell or a B cell.
  • 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 of (i).
  • Embodiment 17 The method of any one of Embodiments 14-16, wherein, in step (c), the second nucleic acid molecule is randomly interrupted by a transposase and a linker sequence is added to its 5' end.
  • the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.
  • Embodiment 18 The method of any one of Embodiments 14-17, wherein the method further comprises step (d):
  • 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.
  • Embodiment 19 The method of embodiment 14, comprising:
  • sequence of the nucleic acid molecule library is obtained.
  • Embodiment 20 The method of Embodiment 19, wherein the method further comprises step (c):
  • 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.
  • Embodiment 21 The method of embodiment 14, comprising:
  • sequence of the nucleic acid molecule library is obtained.
  • 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.
  • step (c) the second nucleic acid molecule a is randomly fragmented by a transposase and a linker sequence is added to its 5' end.
  • the linker sequence comprises the consensus sequence R2 or a partial sequence thereof.
  • 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).
  • the method specifically amplifies and enriches the second nucleic acid molecule a by using a primer G carrying a biotin label.
  • 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.
  • the amplification and enrichment further comprises using a primer H, wherein the primer H comprises a consensus sequence P1 or a partial sequence thereof.
  • Embodiment 24 The method of Embodiment 22 or 23, wherein the method further comprises step (d):
  • 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.
  • Embodiment 25 The method of any one of Embodiments 21-24, wherein the method further comprises step (d)':
  • 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.
  • Embodiment 26 A method for performing omics sequencing on a cell or a cell nucleus, comprising:
  • the nucleic acid molecule library is sequenced.
  • nucleic acid molecule libraries prior to 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
  • Each sub-library 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 the nucleic acid molecules derived from different libraries have different third tag sequences or different fourth tag sequences from each other.
  • Embodiment 27 A nucleic acid molecule library constructed by the method described in any one of embodiments 14-25.
  • Embodiment 28 A reagent composition having the characteristics selected from I, II and III:
  • the reagent composition comprises a second oligonucleotide molecule a, the sequence of which 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 reagent composition further comprises one or more selected from the following:
  • 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;
  • 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;
  • 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 common sequence R1 or a partial sequence thereof;
  • 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
  • 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
  • the consensus sequence O is identical or partially identical to the consensus sequence T
  • 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;
  • 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;
  • transposase complex II 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; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;
  • 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);
  • 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;
  • the reagent composition further comprises one or more selected from the following:
  • 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;
  • 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;
  • transposase complex I wherein the transposase complex I is as defined in embodiment 9;
  • primer D comprises the consensus sequence P1 or a partial sequence thereof
  • primer E' comprises a consensus sequence P1 and an optional third tag sequence
  • primer F' comprises a consensus sequence P2 and an optional fourth tag sequence
  • 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;
  • the reagent composition further comprises one or more selected from the following:
  • 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;
  • 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 common sequence R1 or a partial sequence thereof;
  • 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
  • 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
  • the consensus sequence O is identical or partially identical to the consensus sequence T
  • transposase complex I wherein the transposase complex I is as defined in embodiment 9;
  • 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 to the consensus sequence T. Same or partly the same;
  • primer D comprises the consensus sequence P1 or a partial sequence thereof
  • 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;
  • 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;
  • 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
  • primer H contains a consensus sequence P1 or a partial sequence thereof
  • the consensus sequence O is identical or partially identical to the consensus sequence T
  • 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; in certain embodiments, the linker sequence comprises a consensus sequence R2 or a partial sequence thereof;
  • 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 some embodiments, the cDNA 3’ terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).
  • Embodiment 29 The reagent composition of Embodiment 28, further comprising a reagent for fixing and/or permeabilizing cells or cell nuclei.
  • the reagent composition further comprises methanol, formaldehyde and/or paraformaldehyde.
  • the reagent composition further comprises Triton X-100, digitonin, IGEPAL (e.g., CA-630), and/or Tween-20.
  • 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.
  • an RNase inhibitor e.g., 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
  • the reagent composition further comprises reagents for sequencing; for example, reagents for next-generation sequencing.
  • Embodiment 30 A kit comprising: a multi-reaction system containing a plurality of oligonucleotide molecules, each of the oligonucleotide molecules containing a specific tag sequence;
  • the oligonucleotide molecules in each reaction system have the same tag sequence, and the oligonucleotide molecules in different reaction systems have different tag sequences.
  • 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 120, at least 200, at least 240, at least 500, at least 96, at least 100, at least 12 ... 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 ) multiple reaction systems containing oligonucleotides;
  • 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 120, at least 200, at least 240, at least 500, at least 96, at least 100, at least 12 ... at least 100, at least 200, at least 384, at least 400, 2-5, 2-10
  • the multi-reaction system is preferably a multi-well plate, and the oligonucleotides can be free or fixed in the reaction system.
  • Embodiment 31 A method for fixing and permeabilizing cells, comprising the following steps:
  • the concentration used is 0.05%-2% (e.g., 0.05%-0.2%, 0.05%-0.25%, 0.05%-0.3%, 0.05%-0.5%, 0.05%-0.8%, 0.05%-1%, 0.1%-0.2%, 0.1%-0.25%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%-0.8%
  • the cells are permeabilized by treating the cells with Triton X-100 (0.1%-1%, 0.2%-0.25%, 0.2%-0.3%, 0.2%-0.4%, 0.2%-0.5%, 0.2%-0.8%, 0.2%-1%, or 0.2%) 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).
  • the cell is a naturally occurring cell or a recombinant cell, or a mixture of both.
  • Embodiment 32 A method for fixing and permeabilizing a cell nucleus, comprising the following steps:
  • the permeabilization solution further comprises IGEPAL (e.g., CA-630) and/or Tween-20.
  • IGEPAL e.g., CA-630
  • Tween-20 e.g., Tween-20
  • 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%).
  • IGEPAL e.g., CA-630
  • 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%.
  • 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%).
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • the term "pseudo-monocell” refers to a situation in which a micro-reaction system (e.g., an oil-in-water droplet or a microwell) contains two or more cells in a transcriptomic experiment analyzing a single cell.
  • a micro-reaction system e.g., an oil-in-water droplet or a microwell
  • two or more cells in the same micro-reaction system e.g., the same droplet or microwell
  • the sequencing data generated by the "pseudo-monocell" micro-reaction system cannot be used to analyze the transcriptome information of a single cell because it contains sequencing results from two or more cells. Therefore, in the traditional high-throughput single-cell transcriptome sequencing method, it is necessary to filter or remove the sequencing data generated by the "pseudo-monocell” micro-reaction system from the final sequencing data; and, in order to avoid a large amount of waste of sequencing data, it is necessary to reduce or control the "pseudo-monocell" micro-reaction system as much as possible.
  • the term “pseudomonas rate” refers to the ratio of "pseudomonas" microreaction systems (number) to all microreaction systems (number) containing cells.
  • 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.
  • 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.
  • 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 immune responses.
  • 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.
  • Different cell/cell nuclei types may include different tissue cells/cell nuclei of an individual or the same tissue cells/cell nuclei of different individuals, or cells/cell nuclei of microorganisms derived from different genera, species, strains, variants, or any or all of the foregoing combinations.
  • different cell/cell nuclei types may include normal cells/cell nuclei and cancer cells/cell nuclei of an individual; various cell/cell nuclei types obtained from human subjects, such as a variety of immune cells/cell nuclei; a variety of different bacterial species, strains, and/or variants from environmental, forensic, microbial groups, or other samples; or any other various mixtures of cell/cell nuclei types.
  • 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 qualitatively and/or quantitatively represents the sequence of the target nucleic acid molecule from which the labeled nucleic acid fragment was generated.
  • discrete partitions refer to mutually independent spatial units containing target substances, such as droplets or holes. 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 allocated according to different needs, such as dyes, emulsifiers, surfactants, stabilizers, polymers, aptamers, reducing agents, initiators, biotin markers, fluorophores, buffers, acidic solutions, alkaline solutions, light-sensitive enzymes, pH-sensitive enzymes, aqueous buffers, detergents, ionic detergents, non-ionic detergents, etc.
  • substances allocated according to different needs such as dyes, emulsifiers, surfactants, stabilizers, polymers, aptamers, reducing agents, initiators, biotin markers, fluorophores, buffers, acidic solutions, alkaline solutions, light-sensitive enzymes, pH-sensitive enzymes, aqueous buffers, detergents, ionic detergents
  • cDNA refers to "complementary DNA” synthesized by extension of a primer annealed to the RNA molecule of interest catalyzed by RNA-dependent DNA polymerase or reverse transcriptase using at least a portion of the RNA molecule of interest as a template (this process is also called “reverse transcription”).
  • the synthesized cDNA molecule is "homologous” or “complementary” or “base paired” or “forms a complex” with at least a portion of the template.
  • 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).
  • a transposon end e.g., a transposon, a transposon end, a transposon end composition
  • a transposon end e.g., a double-stranded nucleic acid molecule
  • a transposition reaction e.g., an in vitro transposition reaction
  • 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 the transposition activity (e.g., having higher transposition activity) of the above transposases.
  • the nucleic acids or polynucleotides of the present invention may include, but is not limited to: (1) alteration of the Tm; (2) alteration of the susceptibility of the polynucleotide to one or more nucleases; (3) provision of a moiety for attachment of a label; (4) provision of a label or label quencher; or (5) provision of a moiety for attachment of another molecule in solution or bound to a surface, such as biotin.
  • the nucleic acid or polynucleotide 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 in the transposase complex, the non-transferred strand
  • 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 the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom;
  • the 3' end of the oligonucleotide can be treated with dideoxy to make the 3' end unable to be extended; in some embodiments, the 5' end of the nucleic acid or polynucleotide of
  • the nucleic acid base in the single nucleotide at one or more positions in the polynucleotide or oligonucleotide 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
  • nucleic acid bases 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.
  • 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 in the transposase complex, the non-transferred strand
  • one or more of the sugar moieties may include 2'-deoxyribose, or alternatively, one or more of the sugar moieties may include some other sugar moiety, such as, but not limited to: ribose or 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose that provide resistance to some nu
  • 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 include modified linkages such as, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.
  • the first tag sequence, the second tag sequence, the third tag sequence, the fourth tag sequence, Unique molecular tag sequence, tag sequence is not limited by its composition or length, as long as it can play a role in identification.
  • 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.
  • 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.
  • 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 chain, non-transferred chain 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 its composition of the sequence for various reasons, which will not be repeated here.
  • the consensus sequence R1 is the same as the Read1 sequence of the 10X Genomics platform or a partial sequence thereof or a complementary sequence thereof.
  • the consensus sequence R2 is the same as the Read2 sequence of the 10X Genomics platform or a partial sequence thereof or a complementary sequence thereof.
  • the consensus sequence O is the same as the TSO sequence of the 10X Genomics platform or a partial sequence thereof or a complementary sequence thereof.
  • the consensus sequence P1 is the same as the P5 sequence of the 10X Genomics platform or a partial sequence thereof or a complementary sequence thereof.
  • the consensus sequence P2 is the same as the P7 sequence of the 10X Genomics platform or a partial sequence thereof or a complementary sequence thereof.
  • a bead generally refers to a particle.
  • a bead may be porous, non-porous, solid, semi-solid, semi-fluid or fluid.
  • a bead may be magnetic or non-magnetic.
  • a bead may be soluble, rupturable or degradable.
  • a bead may be non-degradable.
  • a bead may be a gel bead.
  • a gel bead may be a hydrogel bead.
  • a gel bead may be formed from a molecular precursor, such as a polymer or a monomeric substance.
  • a semi-solid bead may be a liposomal bead.
  • the present invention can simultaneously meet the following five points:
  • the empty rate of the micro-reactor system is greatly reduced, which can increase the cell throughput of the existing single-cell omics library construction system based on the micro-reactor system by 10-100 times, reaching 100,000-1 million cells per reaction;
  • the data quality obtained by this scheme is close to that obtained by standard operation of the commercial 10x Genomics platform.
  • the indicators include but are not limited to: number of genes detected, VDJ capture rate, and detected ATAC-seq signal.
  • Figure 1 Existing 10X Genomics single-cell 3'RNA-Seq exemplary library construction process principle and library structure. Specifically, single-cell suspension, reverse transcription reaction solution and 10X genomics cell barcode-labeled microbeads are prepared into oil-in-water microdroplets with one cell plus one magnetic bead on the 10X genomics platform. After the microdroplets are collected and reverse transcribed and template replaced on the PCR instrument, the cell barcode on the microbead will be loaded into the cell's cDNA product, and the final library will be obtained after subsequent cDNA amplification, enzyme cutting and adding adapters, and PCR amplification.
  • 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; the UMI (unique molecular identifiers) single molecule barcode is used to mark different mRNAs in the same cell; Poly (dT) is a polythymine oligonucleotide, which is introduced during the reverse transcription process of mRNA; the thin line in the middle is the transcriptome sequence.
  • FIG 2 Schematic diagram of the exemplary process principle of the present invention.
  • the present invention is based on the cell barcode labeling combined with the post-combination labeling method of micro-reaction (such as the oil-in-water microdroplet shown in Figure 2).
  • the post-combination labeling method of micro-reaction such as the oil-in-water microdroplet shown in Figure 2).
  • the cells are re-mixed and distributed, and the second round of labels (96-384 types) are introduced to the cells on the nucleic acid molecules of the cells, realizing a variety of new single-cell omics library construction schemes.
  • Figure 3 Schematic diagram of exemplary single-cell transcriptome, VDJ library construction and library structure of the present invention.
  • the present invention prepares oil-in-water microdroplets of intact 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. Through PCR amplification, different second-round labels are added to the cells in different wells. Next, the amplified products are collected.
  • the amplification product of the 5' end RNA of a single cell can be further enriched for VDJ to obtain a VDJ library.
  • the final library structure is: P5 and P7 at the two ends are the sequencing adapter sequences of illumina; i7 on the right is the label sequence of the library, 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 label; the part marked with round 2 is the second round of cell label introduced by index PCR; UMI (unique molecular identifiers) single molecule barcode is used to mark different mRNAs in the same cell; TSO is the template replacement sequence; the unmarked part in the middle is the transcriptome sequence.
  • Figure 4 Schematic diagram of an exemplary single-cell multi-omics library construction and library structure of the present invention.
  • the present invention prepares oil-in-water microdroplets of intact 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 through a reverse transcription reaction (3’RNA)/gDNA in the open region of chromatin through a ligation reaction in situ on 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. Through PCR amplification, different second-round labels are added to the cells in different wells.
  • the amplified products are collected together for purification and further library construction, and finally the two products are enriched by the corresponding primers of 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.
  • FIG5 The results of sequencing mixed samples of human and mouse cell lines using the single-cell transcriptome method of the present invention.
  • FIG6 The results of sequencing human peripheral blood mononuclear cell samples fixed under different conditions using the transcriptome method of the present invention.
  • FIG7 The results of sequencing frozen human peripheral blood mononuclear cell samples using the single-cell 5’ RNA-seq method of the present invention.
  • FIG8 The results of sequencing human peripheral blood mononuclear cell samples using the single-cell VDJ-seq method of the present invention.
  • FIG9 The results of sequencing frozen human kidney samples using the single-cell transcriptome + ATAC multi-omics method of the present invention.
  • micro-reaction cell barcode labeling platforms taking the Chromium platform of 10X Genomics as an example, Not all micro-reaction systems (GEM, water-in-oil droplets) contain single cells as expected. Usually, there will be situations containing two or more cells, which are also called “pseudo-monocellular cells". In the case of "pseudo-monocellular cells", two or more cells in the same GEM will be marked with the same barcode. This results in the inability to perform a "one-to-one" identification of two or more cells present in the GEM using only the barcode in the GEM.
  • the sequencing data generated by the "pseudo-monocellular" GEM cannot be used to analyze the transcriptome information of a single cell because it contains sequencing results derived from two or more cells. Therefore, it is necessary to filter or remove the sequencing data generated by the "pseudo-monocellular" GEM from the sequencing data finally generated; and in order to avoid a large amount of waste of sequencing data, it is necessary to reduce or control the number or ratio of the "pseudo-monocellular" GEM as much as possible, thereby greatly limiting its library construction throughput.
  • the present invention Based on the barcode labeling of cells in micro-reactions (e.g., water-in-oil droplets, nano-micropores), the present invention removes the cells/nuclei that have completed the first round of cell barcode loading in situ in the cells/nuclei in the micro-reaction system, mixes them thoroughly and divides them into several equal parts, then introduces the second round of labeling on the nucleic acid molecules of the cells/nuclei through the index PCR of the micro-system, and finally uses the two rounds of label information to jointly define a cell.
  • micro-reactions e.g., water-in-oil droplets, nano-micropores
  • 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 the "pseudo-monocytes", and then accurately track and determine the cell source of the sequencing data.
  • two or more cells in a "pseudo-monocyte" micro-reaction system all contain the same first round of cell barcode labels
  • the two or more cells each contain different second round of cell barcode labels, so that the sequencing data generated by each cell therein can be distinguished according to the second round of cell barcode labels, so that even the sequencing data generated by the "pseudo-monocytes" can be used.
  • the applicant also hopes to emphasize that the method based on pre-labeling and microfluidic droplet high-throughput library construction technology can improve the throughput and reduce the empty rate and pseudo-single cell rate of the micro-reaction system compared with the existing traditional microfluidic droplet high-throughput library construction technology.
  • the reagents used in the examples of the present application have the meanings generally understood by those skilled in the art.
  • the reagents used in the examples of the present application can be purchased from the market or prepared by themselves according to the formula widely used in the corresponding field.
  • Example 1 Fixation and permeabilization of single cell suspension
  • the library can be constructed using intact single cells from fresh tissues, fresh cell lines, fresh blood samples, primary cells, and frozen cell samples. Before the library is constructed, the cells need to be fixed and permeabilized.
  • Hela cell line, NIH3T3 cell line (purchased from the cell bank of the Chinese Academy of Sciences) and peripheral blood mononuclear cells PBMC were used for the experiment.
  • fixation and permeabilization steps are as follows:
  • Method 1 Fix and permeabilize in 80% methanol at -20°C for 10 min.
  • 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.
  • 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.
  • the library can be constructed using cell nuclei from fresh tissues, frozen tissues, cell lines, blood samples, primary cells, and frozen cell samples (the extraction method of cell nuclei refers to the conventional steps widely used in the field). Before library construction, cell nuclei need to be fixed and permeabilized.
  • fixation and permeabilization steps are as follows:
  • Method 1 Fix with 1% formaldehyde at room temperature for 10 minutes.
  • Method 2 Fix with 1.6% paraformaldehyde at room temperature for 5 minutes.
  • the single-end (i7-end) TN5 transposase complex will be used in the single-cell transcriptome library construction process of the present invention.
  • Transposon preparation Tn5-top_ME nucleotide (SEQ ID NO: 9) and Tn5-bottom_Read2N nucleotide (SEQ ID NO: 10) were respectively prepared by TruePrep Dissolve the annealing buffer in the Tagment Enzyme kit to 100 Um, and then mix the two nucleotides in a 1:1 volume ratio. In the embodiment of the present invention, take 10ul of the two nucleotides respectively and mix them thoroughly. Place in a PCR instrument and perform the following annealing reaction program: 75°C 15 minutes, 60°C 10 minutes, 50°C 10 minutes, 40°C 10 minutes, 25°C 30 minutes. The annealed adapter mixture is the transposon and is stored at -20°C.
  • TN5 transposase complex embedding using TruePrep The TruePrep Tagment Enzyme (2 ⁇ g/ ⁇ l) and Coupling Buffer in the Tagment Enzyme Kit are used to prepare the following reaction solution: 10ul TruePrep Tagment Enzyme (2 ⁇ g/ ⁇ l), 33ul Coupling Buffer, 7ul transposon (obtained in the previous step). After thorough mixing, place in a PCR instrument and react at 30°C for 1 hour. After the reaction is completed, a single-end (i7-end) TN5 transposase complex is obtained and stored at -20°C.
  • Example 4 Preparation of single-cell transcriptome library (including single-cell 3'RNA-
  • Example 10X genomics chromium platform 10x Single Cell 5'RNA-seq and 10x Single Cell 3'RNA-seq system are used as examples to prepare oil-in-water microdroplets, giving each microdroplet a unique label.
  • the microbeads for oil-in-water preparation and cell barcode labeling can be replaced by other platforms.
  • the 10x Single Cell 5'RNA-seq reaction system is: 18.8 ⁇ l RT Reagent B, 7.3 ⁇ l Poly-dT RT Primer, 1.9 ⁇ l Reducing Agent B, 2 ⁇ l RT Enzyme C, 38.7ul fixed and permeabilized cell/cell nucleus suspension;
  • the 10x Single Cell 3'RNA-seq reaction system is: 18.8 ⁇ l RT Reagent B, 2.4 ⁇ l Template Switch Oligo, 2 ⁇ l Reducing Agent B, 8.7 ⁇ l RT Enzyme C, 43.2ul fixed and permeabilized cell/cell nucleus suspension.
  • the oil-in-water product that has completed the first round of barcode cell label loading breaks the oil-in-water microdroplets, takes out the cells from the aqueous phase and mixes them thoroughly, and then distributes the cell/cell nucleus suspension into a 96-well plate.
  • Cell lysis and purification Place the 96-well plate with cells in a PCR instrument and incubate at 85°C for 5 minutes. Then perform purification.
  • Index PCR amplification reaction (loading the second round of cell labeling): Add cDNA amplification reaction solution to the above 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), 2ul 10uM Truseq-i5-end specific second label primer (sequence as SEQ ID NO: 5, there are 96 types of primers used in this embodiment, one is added to each well, and the label sequences contained in the 96 primers are respectively selected from the sequences shown in SEQ ID NO: 7), mix well and quickly place in a PCR instrument for amplification.
  • This example provides a library construction method in which a single-end transposase is inserted into the i7-end sequencing primer and then amplified.
  • 100 ng of the product from the previous step is taken and interrupted by transposition 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 Index PCR cDNA amplification product, and the reaction solution is fully mixed and placed in a PCR instrument for incubation at 55°C for 15 minutes. Purify and elute with 0.8x SPRIselect magnetic beads.
  • the reaction system was: 50ul NEBNext High-Fidelity 2x PCR Master Mix, 5ul 10uM P5 end primer (SEQ ID NO: 1), Nextare-i7 end second label primer (sequence structure schematic as shown in SEQ ID NO: 6, the label sequence contained in the primer actually used in this embodiment is selected from the sequence shown in SEQ ID NO: 8), 40ul transposition purification product.
  • reaction conditions 72°C 5min, 98°C 45s, 8 cycles [98°C 20s, 60°C 30s, 72°C 1min], 72°C 5min, 4°C temporary storage.
  • Sequencing library purification and fragment screening Use 0.6X and 0.2X SPRIselect magnetic beads to purify and screen the products from the previous step. Finally, a sequencing library with a fragment size of about 300-600bp is obtained.
  • Example 5 Preparation of single cell VDJ library (taking human peripheral blood mononuclear cells as an example)
  • 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 products shown in Example 4 using immune cells T cells and B cells, that is, using the 10X genomics chromium platform 10x Single Cell 5'RNA-seq to complete steps 1 to 5 of Example 4, and performing nested PCR amplification of the obtained cDNA amplification products loaded with two rounds of markers using primers specific for the VDJ conserved region to enrich for the VDJ sequence.
  • the enriched product still carries the same two rounds of cell markers as the single-cell 5'RNA-seq transcriptome.
  • This example takes PBMC from human peripheral blood as an example, and constructs VDJ libraries of T cells and B cells therein respectively.
  • Nested PCR specific enrichment of VDJ sequences After the 10x Single Cell 5' RNA-seq Index PCR cDNA amplification product in Example 4 was purified from PBMCs derived from human peripheral blood, 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 primers (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 should be mixed 1:1 before amplifying TCR/BCR), 5ul cDNA amplification product, 35ul nuclease-free water, mix thoroughly and quickly place in PCR instrument, the reaction conditions are as follows: 98°C 45s, amplify TCR for 11 cycles [98°C 20s, 62°C 30s, 72°C 1min], 72°C 1min, and store at 4°C.
  • the PCR amplification product was purified and fragment screened using 0.5X and 0.3X SPRIselect magnetic beads, and eluted with 40.5ul EB buffer. Then, the second round of nested PCR amplification was carried out.
  • the PCR reaction system was: 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), 7 primers for B Cell (SEQ ID NOs: 22-28), and the corresponding primers were mixed 1:1 before amplifying TCR/BCR), 40ul of the first round of nested PCR amplification product was fully mixed and quickly placed in the PCR instrument.
  • 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 stored at 4°C. Finally, the final PCR amplification product was purified and fragment screened using 0.5X and 0.25X SPRIselect magnetic beads and eluted with 30.5ul EB buffer. 1ul of the eluted product was taken and the concentration was measured using Qubit. The remaining sample can be stored at -80°C for 3 months.
  • VDJ sequencing library Same as Example 4, this example provides a library construction method in which a single-end transposase is transposed and inserted into the i7-end sequencing primer and then amplified. 100ng of VDJ enriched product is taken and interrupted by transposition 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 Index PCR cDNA amplification product, the total reaction system is 50ul, and the insufficient volume is supplemented with nuclease-free water. After the reaction solution is fully mixed, it is placed in a PCR instrument and incubated at 55°C for 5min. The product is purified with 0.8x SPRIselect magnetic beads,
  • the magnetic beads were eluted with 40.5ul EB.
  • the purified product was amplified for sequencing library, and the reaction system was: 50ul NEBNext High-Fidelity 2x PCR Master Mix, 5ul 10uM P5 end primer, 5ul 10uM P5 end primer Nextare-i7 end second label primer (the sequence structure is shown in SEQ ID NO:6, and the label sequence contained in the primer actually used in this embodiment is selected from SEQ ID NO:8), 40ul transposition purification product.
  • reaction conditions 72°C 5min, 98°C 45s, 7 cycles [98°C 20s, 60°C 30s, 72°C 1min], 72°C 5min, 4°C temporary storage.
  • Sequencing library purification and fragment screening Use 0.6X and 0.2X SPRIselect magnetic beads to purify and screen the products from the previous step. Finally, a sequencing library with a fragment size of about 300-600bp is obtained.
  • Example 6 Preparation of single-cell mRNA+genomic DNA multi-omics library (taking human kidney single-cell sample as an example)
  • the embodiment uses the 10X genomics chromium platform Single Cell Multiome ATAC+RNA-seq system to first complete the oil-in-water microdroplet preparation and the first round of cell label barcode loading. On this basis, the second round of cell labels are loaded on the transcriptome and chromatin open area of the cell/nucleus through index PC, and finally the construction of the single-cell multi-omics library is completed.
  • the oil-in-water preparation and cell barcode-labeled microbeads can be replaced by other platforms. Specific method:
  • In situ cell transposition reaction According to the Chromium Next GEM Single Cell Multiome ATAC+Gene Expression User Guide, the fixed and permeabilized single cell nuclei and permeabilized cell samples in the above example were subjected to in situ transposition reaction.
  • the reaction system was: 7ul ATAC Buffer B, 3ul ATAC Enzyme B, 5ul fixed cell/nucleus suspension. After thorough mixing, the suspension was placed in a PCR instrument.
  • the reaction conditions were as follows: 37°C for 60 min, and then temporarily stored at 4°C. After the reaction, a specific linker sequence was introduced into the open chromatin region of the sample cell nucleus.
  • Cell lysis and purification Add 1ul Proteinase K to each well of the above-mentioned products, mix thoroughly and centrifuge, then place in a PCR instrument and incubate at 55°C for 5 minutes. Then configure Dynabeads Cleanup Mix according to the 10X Chromium Single Cell Reagent Kits User Guide, add 16ul Dynabeads Cleanup Mix to each well of the 96-well plate for purification, and finally elute with 16.5ul Elution Solution I to each well, and transfer the eluate to a new 96-well plate. The purified product was purified again with 1.8x SPRIselect magnetic beads, and finally eluted with 16.5ul EB, and the eluted product was transferred to a new 96-well plate again.
  • Index PCR amplification reaction (loading the second round of cell labels): Add cDNA and gDNA amplification reaction solution to the above purified products.
  • the reaction solution to be added to each well of the 96-well plate includes: 25ul NEBNext High-Fidelity 2x PCR Master Mix, 2ul 10uM P5 end primer (SEQ ID NO: 1), 2ul 10uM Nextare-i7 end second label primer (sequence such as SEQ ID NO: 6, there are 96 kinds of primers used in this embodiment, one is added to each well to label the gDNA from the chromatin open region, and the label sequences contained in the 96 primers are selected from SEQ ID NO: 6.
  • Enrichment of cDNA amplification products Take 40ul of the above Index PCR amplification products and enrich the cDNA amplification products with biotin-modified primers, add 60ul reaction solution, the reaction solution contains: 50ul KAPA HiFi HotStart 2X ReadyMix, 5ul 10uM P5 end primer (SEQ ID NO: 1), 5ul 10uM Bio-Partial TSO/IS primer (SEQ ID NO: 3), mix well and quickly place in PCR instrument, the reaction conditions are as follows: 98°C 30s, 6 cycles [98°C 20s, 54°C 30s, 72°C 20s], 72°C 1min, 4°C temporary storage.
  • the C1beads adsorbed with the cDNA amplification product were resuspended in new PCR reaction solution (the reaction solution contained: 50ul KAPA HiFi HotStart 2X ReadyMix, 5ul 10uM P5 end primer (SEQ ID NO: 1), 5ul 10uM Partial TSO/IS primer (SEQ ID NO: 2), 40ul nuclease-free water), and further amplified after thorough mixing.
  • the reaction solution contained: 50ul KAPA HiFi HotStart 2X ReadyMix, 5ul 10uM P5 end primer (SEQ ID NO: 1), 5ul 10uM Partial TSO/IS primer (SEQ ID NO: 2), 40ul nuclease-free water), and further amplified after thorough mixing.
  • the reaction conditions were as follows: 98°C 30s, 4 cycles [98°C 20s, 54°C 30s, 72°C 20s], 72°C 1min, and stored at 4°C.
  • the PCR tube was placed on a magnetic stand for 5min, the supernatant was aspirated, and the supernatant was purified with 0.6x SPRIselect magnetic beads and eluted with EB. Take 1ul of the eluted product and measure the concentration with Qubit. The remaining sample can be stored at -80°C for 3 months.
  • ATAC-seq sequencing library construction Take 40ul of the purified Index PCR amplification product in step 6 of this implementation to construct the ATAC-seq sequencing library. Add 50ul KAPA HiFi HotStart2X ReadyMix, 5ul 10uM P5 end primer (SEQ ID NO: 1), 5ul 10uM P7 end primer (SEQ ID NO: 4) to the 40ul Index PCR amplification product, mix well and then perform PCR amplification. Reaction conditions: 98°C 45s, 7-10 cycles (depending on the number of cells loaded) [98°C 20s, 67°C 30s, 72°C 20s], 72°C 1min, and store at 4°C.
  • ATAC-seq sequencing library purification and fragment screening Use 0.4X and 1X SPRIselect magnetic beads to purify and screen the products from the previous step. Finally, a sequencing library with a fragment size of about 200-700bp was obtained.
  • ATAC-se library sequencing The constructed library was sequenced using NovaSeq 6000 (Illumina, San Diego, CA) with a read length of 50 bp and 25,000 reads per cell.
  • FIG5 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 a scatter plot of the number of UMIs in a single cell mapped to the genomes of different species, wherein the number of UMIs is the number of cDNA molecules sequenced, and each point in the figure represents a cell (a total of 6446 points), wherein light-colored points represent cells that contain almost only mouse cDNA, dark-colored points represent cells that contain almost only human cDNA, and black points represent cells that are contaminated (i.e., pseudomonocytes).
  • 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 is the number of UMIs detected after peripheral blood mononuclear cells were fixed under three conditions (methanol, 1% formaldehyde, 1% paraformaldehyde);
  • B is the number of genes detected after peripheral blood mononuclear cells were fixed under three conditions (methanol, 1% formaldehyde, 1% paraformaldehyde);
  • C is the visualization result of unsupervised clustering of all cells under the three fixing conditions;
  • D is the distribution of cell clusters under the three fixing conditions.
  • 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 27 major cell types in the blood were detected in this method.
  • 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 the visualization results of cells with detected BCR/TCR clones
  • black dots are cells with detected BCR/TCR clones
  • light dots are cells without detected BCR/TCR
  • cells with detected BCR clones completely overlap with the B cell position annotated in the single-cell transcriptome data of Figure 7
  • cells with detected TCR clones completely overlap with the T cell position annotated in the single-cell transcriptome data of Figure 7
  • B and D are the proportions of B cells and T cells with detected BCR and TCR clones, respectively.
  • Figure 9 shows the results of sequencing frozen human kidney samples using the single-cell transcriptome + ATAC multi-omics method of the present invention.
  • A is the cell clustering visualization result of the single-cell transfer group of the frozen human kidney sample, showing that the 12 major cell types in the kidney are detected in this method;
  • B is the number of genes detected in a single cell of the single-cell transcriptome of the frozen human kidney sample;
  • C is the cell clustering visualization result of the single-cell ATAC-seq part of the frozen human kidney sample, and 18 cell clusters are obtained by clustering;
  • D is the ATAC-seq peak information obtained for various cell types in the single-cell ATAC-seq part.

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Abstract

L'invention concerne un procédé d'introduction d'une étiquette spécifique à deux cellules dans des molécules d'acide nucléique dérivées de cellules, et la construction d'une bibliothèque de molécules d'acide nucléique pour le séquençage de transcriptome à cellule unique, le séquençage d'accessibilité de la chromatine à cellule unique ou le séquençage d'accessibilité de la chromatine à cellule unique multi-omique sur la base du procédé, ou un procédé pour effectuer un séquençage à haut débit sur le transcriptome à cellule unique, l'accessibilité de la chromatine à une seule cellule ou l'accessibilité de la chromatine à cellule unique multi-omique. L'invention concerne en outre une bibliothèque de molécules d'acide nucléique construites à l'aide du procédé, et un kit pour mettre en œuvre le procédé.
PCT/CN2023/119479 2023-09-18 2023-09-18 Procédé et kit de marquage à haut rendement de molécules d'acide nucléique cellulaire Pending WO2025059808A1 (fr)

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