WO2024250155A1 - Procédé de construction d'une banque de séquençage de cellules individuelles - Google Patents

Procédé de construction d'une banque de séquençage de cellules individuelles Download PDF

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WO2024250155A1
WO2024250155A1 PCT/CN2023/098377 CN2023098377W WO2024250155A1 WO 2024250155 A1 WO2024250155 A1 WO 2024250155A1 CN 2023098377 W CN2023098377 W CN 2023098377W WO 2024250155 A1 WO2024250155 A1 WO 2024250155A1
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seq
index
cells
vector
linker
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张强锋
唐磊
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Tsinghua University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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

  • the present invention relates to the technical field of gene sequencing, and in particular to a method for pairing analysis of RNA and chromatin accessibility in the same single cell through sequencing.
  • Single-cell sequencing technology has developed from single-cell RNA-seq to ultra-high-throughput, multimodal single-cell sequencing.
  • G&T-seq detects the single-cell genome and transcriptome in the same cell.
  • ScTrio-seq analyzes the relationship between the genome, DNA methylation, and transcriptome of a single mammalian cell, and CITE-seq simultaneously measures epitopes and transcriptomes in a single cell.
  • sci-CAR SNARE-Seq
  • Paired-Seq SHARE-Seq
  • Chromium single-cell multi-omics ATAC+ gene expression kits can locate chromatin and RNA in the same single cell. These methods dissect tissue heterogeneity and reveal relevant epigenomic regulatory elements.
  • sci-CAR barcodes have low binding and high collision rates
  • Paired-Seq has suboptimal labeling and reverse transcription reaction efficiencies when there are too many cells per tube.
  • SHARE Seq requires custom sequencing to read two fragments of the ATAC Seq library, increasing sequencing costs.
  • SNARE-Seq uses the Drop-Seq system to encapsulate labeled cells with DNA barcoded microbeads in nanoliter droplets, resulting in low cell yield (10k per experiment) and a high ratio of more than 2 cells with the same barcoded labeling (11.3%).
  • Each single-cell multi-omics ATAC+ gene expression kit obtained the best joint analysis data, but the cost is high and the throughput is similar to SNARE-Seq.
  • DSC-seq droplet-based single-cell sequencing
  • this application proposes an ultra-high-throughput multimodal single-cell technology that measures gene expression and chromatin accessibility in the same cell in parallel, called (Parallel-seq).
  • the present invention provides a single-cell ultra-high-throughput dual-omics technology (single-cell combined fluid labeling (SCIFI)), which can simultaneously measure the gene expression and chromatin accessibility of the same cell.
  • SIMFI single-cell combined fluid labeling
  • Parallel-Seq only performs four rounds of barcode indexing through one round of ligation reaction and two rounds of amplification reaction
  • Parallel-Split-Seq only performs four rounds of barcode indexing through two rounds of ligation reaction and one round of amplification reaction, which realizes the joint analysis of open chromatin and gene expression in the same single cell, and can deconvolve cis-regulatory elements that regulate gene expression.
  • a method for constructing a single-cell sequencing library comprising using a transposon to cut open chromatin to obtain a DNA fragment carrying a first connector; adding a reverse transcription primer to reverse transcribe mRNA to obtain a first chain of cDNA carrying a second connector, thereby constructing a chromatin DNA library and a transcriptome library in the same cell.
  • the method further comprises placing the cells on a vector, and using a first vector-specific linker to connect the DNA fragment carrying the first linker obtained above and the first chain of cDNA obtained above to the vector respectively.
  • the method further comprises synthesizing a second strand of cDNA.
  • the method further comprises forming droplets, lysing cells and performing an amplification reaction in the droplets, and preferably, the formed droplets are overloaded with cells.
  • the method further comprises purifying DNA and amplifying cDNA and chromatin DNA of the transcriptome library using primers, respectively.
  • the method further comprises adding RNase.
  • the method further comprises obtaining cells, fixing and permeabilizing the cells.
  • a method for constructing a single-cell sequencing library comprises:
  • step c) placing the cells on a vector, and using a first vector-specific linker to connect the DNA fragment carrying the first linker obtained in step a) and the first chain of the cDNA obtained in step b) to the vector respectively;
  • step a) and b) can be performed simultaneously or sequentially.
  • step a) can be performed first and then step b), or step b) can be performed first and then step a).
  • step a) is performed first and then step b).
  • it contains more than 10 transposons, more than 100 transposons, more than 1000 transposons, more than 10000 transposons, and so on.
  • the transposon comprises a barcode sequence and a transposase.
  • the transposase includes but is not limited to Tn5 transposase, Mu transposase, Tn7 transposase or IS5 transposase.
  • the transposase is Tn5 transposase.
  • the Tn5 transposase carries a sequence as shown in SEQ ID NO: 1 or 12.
  • the barcode sequence comprises a first adapter. Further preferably, the barcode sequence comprises a first index.
  • the first adapter comprises a first index and a transposase binding site.
  • the first linker comprises at least one linker that is the same or different. Further preferably, the first linker comprises at least 4 linkers that are the same or different. In a specific embodiment of the present invention, it comprises at least 4-96 linkers that are the same or different.
  • the barcode sequence is sequentially composed of an overhang, a first index and a transposase binding site from 5′ to 3′, and the overhang is a sequence complementary to a subsequent primer.
  • the second linker comprises at least one linker that is the same or different. Further preferably, the second linker comprises at least 4 linkers that are the same or different.
  • the reverse transcription primer comprises a second linker, wherein the second linker comprises poly(T) and a first index; and preferably further comprises a random hexamer primer.
  • the reverse transcription primer comprises poly(T) and sequences complementary to the first index and subsequent primers.
  • the first adapter and the second adapter may contain the same sequence complementary to the subsequent primer.
  • the first index comprises at least one of AACAAC, ACCGCA, AGTTGG, CCACGT, CGTGTT, GTTCTC, TGACTA, TCAAGG, AACGGT, AAGCCT, ACATGA, ACTCTA, AGAAGT, AGTACC, ATGCGA, CAATAG, CATCCA, CCTGGA, CGAGAC, CGCTCA, GCGTAA, GGATCG, GTGAGG, TCCTTA, TCTGCC, TTAACC or TTAGTG, or a combination of two or more than three of them.
  • the barcode sequence comprises at least one, two or more combinations of SEQ ID NO: 2 hybridized with SEQ ID NO: 1 or 12 respectively.
  • the reverse transcription primer comprises at least one of SEQ ID NO: 3, 4, or a combination of two or more than three.
  • the first vector-specific adapter comprises a second index.
  • the first vector-specific adapter comprises a UMI.
  • the second index comprises AAGACCAA, AAGCTACG, AAGGTCAT, AATAGTGG, AATGCCCTT, ACAATAGC, ACAGGATT, ACCGACCT, ACCTAGAT, ACGAGTCC, ACGGACGA, ACGTTCAA, ACTATCTG, ACTCCGAA, AGAACAGA, AGACGCTT, AGATGCGA, AGCCACTC, AGCGAAGC, AGGTAACG, AGTACATC, AGTGATTC, ATAAGAGG, ATA TCACG, ATCGCCGT, ATGACGGA, ATGGAATG, ATTCCTAC, CAACGCCA, CAAGTCTG, CACACATC, CACCTTAT, CAGAACCT, CAGCCGAT, CATACTGT, CATCCAC C.
  • the first vector-specific adapter comprises a second index, a UMI, and a sequence complementary to a reverse transcription primer or a transposon sequence.
  • the first vector-specific adapter is composed of, from 5′ to 3′, a sequence complementary to a reverse transcription primer or a transposon sequence, a UMI, a second index, and a sequence complementary to a sequence contained on the vector.
  • the first vector-specific linker comprises SEQ ID NO: 6.
  • the vector contains SEQ ID NO: 5.
  • the first vector-specific linker comprises SEQ ID NO: 15.
  • the vector contains SEQ ID NO: 13.
  • the method also includes the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets, preferably, the formed droplets are overloaded with cells. Overloading the droplets so that all functional droplets are used greatly improves the throughput of the microfluidic device. Linear amplification in droplets avoids the purification of unamplified products and can be easily combined with CRISPR screening, DNA methylation analysis, and protein expression analysis, which may lead to single-cell cross-omics sequencing or even whole-omics sequencing of single cells.
  • the primers used in the amplification reaction in the droplet include a third index.
  • the primer used for the amplification reaction in the droplet comprises SEQ ID NO: 8.
  • the step of lysing the droplets is further included after the linear amplification.
  • the lysed droplets are lysed by using a demulsifier.
  • the method comprises using a second vector-specific adapter to connect the DNA fragment carrying the first adapter and the first strand of cDNA obtained above to a vector, respectively.
  • the second vector-specific adapter comprises a third index.
  • the second vector-specific linker comprises SEQ ID NO: 16.
  • the vector contains SEQ ID NO: 14.
  • the third index comprises AACCTCTT, AACGTCCGC, AAGAATCG, AAGCGGTG, AAGGAGCT, AATACCGC, AATCTCCA, ACAACTTC, ACACGCAA, ACCACAGT, ACCGTGTA, ACCTTGCC, ACGCATAA, ACGTATGG, ACTAACCA, ACTCAGGT, ACTGTTG, AGAAGTAC, AGAGATGA, AGATTAGG, AGCCTGGT, AGCTCTAA, AGGT GTCT, AGTCCGTT, AGTTCGCA, ATAAGCTC, ATCCATGA, ATCTAGCG, ATGCAACC, ATGTGCAG, ATTGGTAG, CAAGAAGA, CAATGGAC, CACATGCT, CACGGTAG, CAGAGGTT, CAGTATAG, CATCAAGT, CAGTTCC, CCAACAAT, CCAATTAC, CCAGTGAA, CCGATCAG, CCGGTCTT, CGACAACG,
  • the method further comprises the step of purifying DNA.
  • the primers used in the amplification reaction performed after the DNA purification comprise a fourth index.
  • the fourth index comprises a combination of at least one, two or more than three of the P3xx indexes;
  • the fourth index includes a combination of at least one, two or more than three of N7xx;
  • the fourth index comprises a combination of at least one, two or more than three of P5xx;
  • the fourth index includes a combination of at least one, two or more than three of N5xx.
  • the primers used to amplify the transcriptome are SEQ ID NO: 9, 10.
  • the primers used to amplify the transcriptome are SEQ ID NO: 20, 18.
  • the primers used to amplify the open chromatin fragments are SEQ ID NO: 9, 11.
  • the primers used to amplify the open chromatin fragments are SEQ ID NO: 20, 19.
  • the carrier comprises a well, a tube or a plate.
  • the carrier is an ELISA plate such as a 96-well plate.
  • the method further comprises adding RNase.
  • RNA is removed from the first-strand cDNA by RNase digestion reaction, and then the second-strand synthesis is performed using random primers, thereby avoiding the destruction of the open chromatin fragments by 0.1N NaOH and the contamination of the RNA-seq library.
  • the method further comprises obtaining cells, fixing and permeabilizing the cells.
  • a method for constructing a multi-mode single-cell sequencing library comprises the method for constructing a single-cell sequencing library according to the above-mentioned method.
  • the third aspect of the present invention provides a method for constructing a transcriptome library, which comprises adding a reverse transcription primer to reverse transcribe mRNA to obtain a first chain of cDNA carrying a second linker; placing cells on a vector, and connecting the obtained first chain of cDNA to the vector using a first vector-specific linker; synthesizing the second chain of cDNA; and purifying and amplifying the cDNA of the transcriptome with primers.
  • the reverse transcription primer comprises a second linker, wherein the second linker comprises poly(T) and a first index; and preferably further comprises a random hexamer primer.
  • the first vector-specific linker comprises a second index.
  • the method further comprises the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets.
  • the formed droplets are overloaded with cells
  • the primers used in the amplification reaction in the droplet include a third index.
  • the method comprises connecting the obtained first-strand cDNA to the vector using a second vector-specific adapter, and preferably, the second vector-specific adapter comprises a third index.
  • the primers used in the amplification reaction performed after the DNA purification comprise a fourth index.
  • the method further comprises adding RNase.
  • the fourth aspect of the present invention provides a method for constructing a chromatin DNA library, which comprises using a transposon to cut open chromatin to obtain a DNA fragment carrying a first linker; placing cells on a vector, and connecting the obtained DNA fragment carrying the first linker to the vector using a first vector-specific linker; purifying the DNA and amplifying the chromatin DNA respectively using primers.
  • the transposon comprises a barcode sequence and a transposase; preferably, the barcode sequence comprises a first linker; further preferably, the barcode sequence further comprises a first index.
  • the first vector-specific linker comprises a second index.
  • the method further comprises the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets.
  • the formed droplets are overloaded with cells
  • the primers used in the amplification reaction in the droplet include a third index.
  • the method comprises connecting the obtained DNA fragment carrying the first linker to the vector using a second vector-specific linker, and preferably, the second vector-specific linker comprises a third index.
  • the primers used to amplify the chromatin DNA comprise a fourth index.
  • the fifth aspect of the present invention provides a nucleic acid library obtained by the above method.
  • the present invention provides a nucleic acid library, wherein the nucleic acid library comprises at least one DNA fragment, and the DNA fragment comprises at least one index and at least one unique molecular identifier.
  • the indexes are one, two, three, four, five, six, seven, eight, nine or more than ten.
  • the index includes a first index, a second index, a third index and/or a fourth index.
  • the nucleic acid library comprises at least one from 5′ to 3′, which is a fourth index, a fragment DNA, a first index, a second index, and a third index.
  • the unique molecular identifier is located between the fourth index and the fragment DNA, between the fragment DNA and the first index, between the first index and the second index, or between the second index and the third index.
  • the seventh aspect of the present invention provides a sequencing method, which comprises constructing the above-mentioned nucleic acid library.
  • the eighth aspect of the present invention provides an application of the above-mentioned nucleic acid library, wherein the application includes tumor target screening, disease monitoring or pre-implantation embryo diagnosis.
  • the ninth aspect of the present invention provides a method for analyzing chromatin accessibility and transcription in the same cell, wherein the method comprises the steps of constructing a single-cell sequencing library, constructing a transcriptome library, and constructing a chromatin DNA library.
  • the tenth aspect of the present invention provides a single-cell multi-omics analysis method, which includes constructing a single-cell sequencing library, constructing a transcriptome library, constructing a chromatin DNA library, and sequencing to obtain chromatin accessibility and/or transcriptome sequence information, and then performing bioinformatics analysis.
  • the eleventh aspect of the present invention provides a kit, which includes reagents used to construct the above-mentioned nucleic acid library.
  • chromatin accessibility refers to the degree of openness of eukaryotic chromatin DNA to other proteins after nucleosomes or transcription factors and other proteins bind to it. Among them, the region that can be re-bound to other proteins is open chromatin.
  • the “carrier” of the present invention can be any object having a solid support surface, and its surface can be modified to couple with cells or nucleic acid molecules. It can be porous glass (CPG), oxalyl-adjusted pore glass, TentaGel support-an amino polyethylene glycol derivatized support, polystyrene, Poros (a copolymer of polystyrene/divinylbenzene) or reversibly cross-linked acrylamide. Many other solid supports are commercially available and suitable for the present invention. In some embodiments, it can be polystyrene resin or poly (methyl methacrylate) (PMMA). It can also be a metal.
  • CPG porous glass
  • PMMA poly (methyl methacrylate)
  • the "droplets" of the present invention are oil-in-water or water-in-oil structures. Different droplets may have different identifiers.
  • the aqueous mixture is combined with an oil phase.
  • the oil phase is a surfactant.
  • the "permeabilization” mentioned in the present invention refers to the technology of changing the permeability of the cell wall and cell membrane without causing cell lysis and destroying the internal organic structure of the cell, so that small molecules and some larger molecules can freely enter and exit the cell. After the permeabilization treatment, the permeability of the cell is improved while the overall structure remains intact, which still has a considerable protective effect on the intracellular enzyme, can ensure that the catalytic effect of the intracellular enzyme is fully exerted, and prolong the service life of the enzyme.
  • the "overload” mentioned in the present invention means exceeding the original carrying capacity.
  • the original carrying capacity is the conventional carrying capacity in the prior art.
  • "overloaded cells in a droplet” means exceeding the amount of cells carried in the original droplet.
  • droplet-carrying cells include empty cells, cells carrying a single cell, or overloaded cells.
  • overloaded cells mean that the number of cells carried in a droplet exceeds one. Preferably, two, three, four, five, six, seven, eight or nine or more cells are carried.
  • the "connector" described in the present invention can be used interchangeably with the adapter in the prior art, and can be used to connect fragmented DNA with an index, or to connect an index with an index, or to connect fragmented DNA with fragmented DNA. It is preferably a nucleotide sequence with a length of 3-1000 bases.
  • index described in the present invention can be used interchangeably with index, barcode, etc. in the prior art.
  • the index can be a sequence or a combination of several sequences. It is preferably a nucleotide sequence with a length of 3-1000 bases.
  • the "unique molecular identifier" mentioned in the present invention is a Unique Molecular Identifier, or UMI for short, which is a randomly designed nucleotide sequence that can specifically identify the molecules it is coupled to. However, not all coupled molecules have a unique UMI. In a specific embodiment, it is combined with other indexes to form a unique molecular identifier.
  • UMI Unique Molecular Identifier
  • Complementarity refers to nucleotide sequences that are related by the base pairing rules.
  • sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'.
  • Complementarity can be partial or complete. Partial complementarity occurs when one or more nucleic acid bases do not match according to the base pairing rules. Complete or complete complementarity between nucleic acids occurs when each nucleic acid base matches another base under the base pairing rules. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and strength of hybridization between nucleic acid chains.
  • the "single cell” mentioned in the present invention refers to a single cell or a cell, which can come from a blood sample, a cell culture, or a specific tissue, organ or tumor, etc. Then, it is separated into single cells by conventional separation methods in the prior art.
  • doublet or “doublets” mentioned in the present invention refers to the situation where two or more cells share a common identifier, such as an index, a linker, a unique molecular identifier, etc. or a combination thereof.
  • nucleic acid refers to DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs and any chemical modifications thereof. Modifications include, but are not limited to, those modifications of chemical groups that provide for integration into other charges, polarizability, hydrogen bonding, electrostatic interactions, connection points and action points with nucleic acid ligand bases or nucleic acid ligands as a whole.
  • Such modifications include, but are not limited to, peptide nucleic acids (PNA), phosphodiester group modifications (e.g., phosphorothioate, methylphosphonate), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitutions of 4-thiouridine, substitutions of 5-bromo or 5-iodo-uracil, backbone modifications, methylation, unusual base pairing combinations such as iso bases, isocytidine and isoguanidine, etc.
  • Nucleic acids may also contain non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, including but not limited to capping with fluorophores (e.g., quantum dots) or other moieties.
  • the terms “comprising” or “including” described in the present invention are open-ended terms.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.
  • the second strand synthesis step in the cell is added to reduce the effect of cross-linked protein inhibition and capture more transcripts.
  • Linear amplification based on droplet indexing is achieved and the efficiency of cDNA capture is improved.
  • a PCR anchor adapter is provided for cDNA that is different from chromatin fragments to avoid ATAC-seq library contamination of RNA-seq library.
  • Parallel-seq overloads droplets with multiple cells to fully utilize the generated droplets, and performs forward and backward indexing to distinguish cells within a droplet, greatly expanding the barcode space. Moreover, the length of the barcode region is significantly reduced, allowing it to read open fragments within the 150nt sequencing read length through the barcode and fixed nucleotide regions.
  • Parallel-Seq first hashes cells with sample-specific barcodes during transposition and reverse transcription, allowing it to evaluate multiple samples in parallel in one experiment and be scalable.
  • Parallel-Seq outperforms existing methods in data quality and has increased throughput (36 million cells per experiment), which provides a powerful tool for building affordable large-scale cell maps.
  • Parallel-seq can easily handle more samples in an experiment and can be expanded to other omics such as DNA methylation, protein expression, and CRISPR screening.
  • Figure 1 Parallel-Seq experimental design diagram, using indexing and droplet overloading to analyze scATAC and scRNA in the same cell, where pool/split represents mixing/dispersion.
  • Figure 2 Parallel-Seq was performed using a mixture of NIH/3T3 (mouse), HEK293T (human), and K562 (human) cells, and the results are mapped to the UMI counts of scRNA-seq (top) and scATAC-seq (bottom) of the human and mouse genomes, where mm10 represents the mm10 version of the mouse reference genome.
  • Figure 3 Insert length distribution of the scATAC-seq subset of Parallel-Seq.
  • Figure 5 Scatter plot showing the log 2 (count) correlation between scATAC-seq and ENCODE DNase-seq by Parallel-Seq in K562 cells.
  • Figure 6 Scatter plot showing the log 2 (TPM+1) correlation between clustered scRNA-seq and ENCODE nuclear RNA-seq in K562 cells Parallel-Seq.
  • Figure 7 Comparison of chromatin accessibility captured by Parallel-Seq and ENCODE DNase-seq, and RNA captured by Parallel-Seq and ENCODE RNA-seq in K562 cells.
  • Figure 8 Uniform Manifold Approximation and Projection (UMAP) visualization of Parallel-Seq paired gene expression data from a mixture of 3T3, 293T, and K562 cells.
  • UMAP Uniform Manifold Approximation and Projection
  • Figure 9 Uniform Manifold Approximation and Projection (UMAP) visualization of Parallel-Seq paired chromatin accessibility data from a mixture of 3T3, 293T, and K562 cells.
  • UMAP Uniform Manifold Approximation and Projection
  • Figure 10 Box plots show the number of uniquely mapped RNA reads and the number of uniquely mapped ATAC reads for sci-CAR, SNARE-seq, Paired-Seq, SHARE-seq, and Parallel-Seq.
  • the horizontal axis RNA library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, and Parallel-Seq from left to right
  • the ATAC library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, and Parallel-Seq from left to right.
  • Figure 11 The box plot shows the number of genes captured per cell in sci-CAR, SNARE-seq, Paired-Seq, SHARE-seq, and Parallel-Seq.
  • the horizontal axis RNA library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, and Parallel-Seq from left to right.
  • Figure 12 Schematic diagram of the Parallel-Split-Seq workflow.
  • FIG. 13 UMI counts for scRNA-seq (left) and scATAC-seq (right) mapped to the human and mouse genomes. This experiment was performed using a mixture of NIH/3T3 (mouse), HEK293T (human), HeLa (human), K562 (human), and THP1 (human) cells for Parallel-Split-Seq.
  • Figure 14 Insert length distribution of scATAC-seq fragments in Parallel-Split-Seq and Parallel-Seq.
  • Figure 15 Enrichment of scATAC-seq reads around TSSs in Parallel-Split-Seq and Parallel-Seq.
  • Figure 16 Scatter plots showing the correlation of log 2 (TPM+1) between scRNA-seq and ENCODE nuclear RNA-seq from Parallel-Split-Seq in K562 cells (Panel A) and the correlation of log2(count) between scATAC-seq and ENCODE nuclear DNase-seq (Panel B).
  • Figure 17 Uniform Manifold Approximation and Projection (UMAP) visualization of Parallel-Split-Seq paired gene expression (left) and chromatin accessibility (right) data from NIH/3T3, HEK293T, HeLa, K562, and THP1 cells.
  • UMAP Uniform Manifold Approximation and Projection
  • Figure 18 Comparative results of chromatin accessibility captured by Parallel-Seq, Parallel-Split-Seq and ENCODE DNase-seq in K562 cells, and comparative results of RNA captured by Parallel-Seq, Parallel-Split-Seq and ENCODE RNA-seq.
  • Figure 19 Box plots show the number of uniquely mapped RNA reads and the number of uniquely mapped ATAC reads for sci-CAR, SNARE-seq, Paired-Seq, SHARE-seq, Parallel-Seq, and Parallel-Split-seq.
  • the horizontal axis RNA library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, Parallel-Seq, and Parallel-Split-Seq from left to right
  • the ATAC library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, Parallel-Seq, and Parallel-Split-Seq from left to right.
  • Figure 20 The box plot shows the number of genes captured per cell in sci-CAR, SNARE-seq, Paired-Seq, SHARE-seq, Parallel-Seq, and Parallel-Split-seq.
  • the horizontal axis RNA library box diagrams are sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, Parallel-Seq, and Parallel-Split-Seq from left to right.
  • HEK293T, HeLa-S3 and NIH/3T3 cells were cultured in DMEM (C11995500BT, ThermoFisher) medium supplemented with 10% fetal bovine serum (P30-3302, PAN BIOTECH) at 37°C and 5% CO 2.
  • the cells were rinsed with PBS (C10010500BT, ThermoFisher) and cultured with 1 mL 0.25% trypsin EDTA (25200114, ThermoFisher) at 37°C for 3-5 minutes to detach the cells.
  • K562 cells were cultured in RPMI 1640 (C11875500BT, ThermoFisher) medium supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 . Detached HEK293T, HeLa-S3 and NIH/3T3 cells and K562 cell suspensions were collected by centrifugation, washed with PBS and counted using Countstar.
  • the tissue was minced into small pieces less than 0.4 mm in a 1.5 mL Eppendorf microcentrifuge tube with scissors.
  • the dissociation mixture was incubated at 37°C and rotated horizontally at 90 rpm for 60 minutes.
  • the single cell suspension was filtered through a 70 ⁇ m cell strainer (15-1070, BIOLOGIX) and centrifuged at 500 g (centrifugal force) for 5 minutes at 4°C.
  • the cells were resuspended with 1 mL PBS and 3 mL red blood cell lysis buffer (4992957, TIANGEN). Incubate at room temperature for 5 minutes and centrifuge at 500 g for 5 minutes at 4°C.
  • the cells were resuspended in 500 ⁇ L fetal bovine serum. 5 ⁇ L of cells were taken, mixed with 5 ⁇ L of Taiban blue solution (15250061, ThermoFisher), and counted with a C-Chip disposable hemacytometer (DHC-N01N, As One). Single cell suspension was diluted to a final concentration of 10% with fetal bovine serum supplemented with dimethyl sulfoxide (D2650, Sigma Aldrich). We cryopreserved cells, with each tube containing 1x10 ⁇ 6 single cells. Before the experiment, cells were gently thawed at 37°C for 5 min and centrifuged at 500g for 5 min at 4°C.
  • Calcein-AM-positive, 7-AAD-negative, and Annexin V-negative single cells were sorted using MoFloAstrios EQ Cell Sorter (Beckman Coulter). Since tumor cells and T cells are likely to be the major part of the sequencing data, we balanced the samples with less than 5% EpCam-positive cells, 40% T cells, and 55% other single cells.
  • Tn5Merev /5Phos/CTGTCTCTTATACACATCT (SEQ ID NO: 21)
  • Tn5ME-A, Tn5ME-B and barcoded R1BxME (x represents 1-96) were prepared.
  • 10 ⁇ M Tn5Merev, 10 ⁇ M Tn5ME-A (for Parallel-Split-Seq) or 10 ⁇ M Tn5ME-B (for Parallel-Seq) were annealed with 10 ⁇ M R1BxME at 95°C for 2 minutes and gradually dropped to 20°C and 4°C at 0.1°C/s.
  • Count 50k single cells for cell lines or classify 50k primary cells for each tube of lung cancer sample Centrifuge single cells at 500g for 5min at 4°C and resuspend single cells in 250 ⁇ L PBS. Add 750 ⁇ L PBS containing 1.33% methanol-free formaldehyde (28906, ThermoFisher) and incubate in ice for 10 minutes. Add 50 ⁇ L 20% BSA (V0332-100G, VWR) and centrifuge at 1000g for 3 minutes at 4°C using swinging bucket centrifugation, then collect cells into 1.5mL microcentrifuge tubes (MCT-150-C, Axygen) and the supernatant was removed in two pipetting steps like the omni ATAC. The results showed that after adding BSA, more primary cells could be recovered by first isolating single cells by swinging bucket centrifugation.
  • 2x RSB was prepared as Omni ATAC by mixing 1 mL 1M Tris HCl pH 7.4 (T2663-1L, Sigma-Aldrich), 200 ⁇ L 5M NaCl (AM9759, ThermoFisher), 300 ⁇ L 1M MgCl 2 (AM9530G, ThermoFisher), and 48.5 mL ultrapure DNase/RNase free distilled water.
  • permeabilization buffer prepare 50 ⁇ L 2x RSB, 1 ⁇ L RiboLock (EO0384, ThermoFisher), 1 ⁇ L SUPERase ⁇ In RNase inhibitor (AM2696, ThermoFisher), 1 ⁇ L 10% Nonidet P40 substitute (1133247301, Sigma-Aldrich), 1 ⁇ L 10% tween20 (11332465001, Sigma-Aldrich), 1 ⁇ L 1% Digitonin (D141-100MG, Sigma-Aldrich), 5 ⁇ L 20% BSA, 40 ⁇ L ultrapure DNase/RNase free distilled water per sample.
  • ATAC-seq reaction solution by mixing 10 ⁇ L 5xLM buffer (M0221, Robustnique), 16.5 ⁇ L PBS, 0.5 ⁇ L RiboLock, 0.5 ⁇ L SUPERase ⁇ In RNase Inhibitor, 0.5 ⁇ L 10% Tween 20, 0.5 ⁇ L Digiton.5 ⁇ L, and 17.5 ⁇ L ultrapure DNase/RNase free distilled water. Resuspend permeabilized single cells with 46 ⁇ L ATAC-seq reaction solution and add 4 ⁇ L barcode-specific transposon to each tube. ATAC-seq reactions were performed at 37°C and 550 r.p.m. with a heated lid.
  • Parallel Seq uses a ligation reaction to add a second index.
  • the ligation adapter contains 7nt complementary strands ligated to the transposon and reverse transcription primers, respectively, as well as a 10nt index strand, an 8nt well-specific adapter, a 10nt UMI, and a universal PCR anchor for droplet linear amplification.
  • the ligation adapter Prior to intracellular barcode ligation, the ligation adapter was annealed by combining 11 ⁇ M of the ligation strand and 12 ⁇ M of the barcode strand in a 100 ⁇ L reaction volume. The plate was incubated at 95°C for 2 minutes and cooled to 20°C at a rate of -0.1°C per second, and the culture plate was then divided into 10 ligation plates, with each well containing 10 ⁇ L of ligation adapter.
  • the second and third indexes were added by ligation reactions.
  • the ligation adapters contained a 10nt sequence complementary to the adapter strand, an 8nt well-specific adapter, and a 7nt sequence, which were then ligated.
  • the ligation adapters added to the ligation reaction for the third index contained a 10nt index strand, an 8nt well-specific adapter, a 10nt UMI, and a short P3 sequence of the universal PCR primer.
  • the second and third round adapters were annealed according to the Parallel-Split-seq protocol and were divided into 10 ligation plates respectively.
  • the intracellular connection steps are as follows:
  • Ligation reactions were performed according to the Split-seq protocol without RNase inhibitors. Prepare 2 mL 1x NEBuffe 3.1 (B7203S, NEB) and 2 mL ligation solution (500 ⁇ L 10x T4 DNA ligation buffer, 100 ⁇ L T4 DNA ligase (M0082, Robustnique), 50 ⁇ L 10% Triton x-100 and 1350 ⁇ L ultrapure DNase/RNase free distilled water). Resuspend the combined single cells with 1x buffer 3.1 and mix thoroughly with the ligation solution. Add 40 ⁇ L of cells from the ligation mixture to each well of the ligation plate. The ligation reaction was rotated at 15 r.p.m for 1 hour at room temperature.
  • Resuspend cells using RNase digestion reaction (40 ⁇ L 5xRT buffer, 8 ⁇ L RNase Cocktail Enzyme Mix (AM2286, ThermoFisher), 8 ⁇ L RNAse H (Y9220L, Enzymatics) and 144 ⁇ L UltraPure DNase/RNase-free distilled water) and incubate at 37°C for 30 min, 300 rpm for 15 s and place on a mixer for 45 s. Wash the RNase digestion reaction by adding 790 ⁇ L PBS and 10 ⁇ L 10% Triton X-100, centrifuge and remove the supernatant. Do not add BSA in this step. Residual BSA will produce fragments with PEG8000 in the next step.
  • RNase digestion reaction 40 ⁇ L 5xRT buffer, 8 ⁇ L RNase Cocktail Enzyme Mix (AM2286, ThermoFisher), 8 ⁇ L RNAse H (Y9220L, Enzymatics) and 144 ⁇ L UltraPure DNase/
  • the second-strand synthesis reaction mixture 40 ⁇ L 5xRT buffer, 48 ⁇ L 50% PEG 8000 (B1004SVIAL, NEB), 20 ⁇ L 10 mM dNTPs, 2 ⁇ L 1 mM dN-P3 short primer (for Parallel-Seq) or dN-P5 short primer (for Parallel-Split-Seq), 5 ⁇ L Klenow Exo- (M0212L, NEB) and 85 ⁇ L UltraPure DNase/RNase-Free distilled water) at 37°C for 1 hour, at 300 r.p.m for 15 s and then on a mixer for 45 s.
  • the second-strand synthesis reaction mixture 40 ⁇ L 5xRT buffer, 48 ⁇ L 50% PEG 8000 (B1004SVIAL, NEB), 20 ⁇ L 10 mM dNTPs, 2 ⁇ L 1 mM dN-P3 short primer (for Parallel-Seq) or dN-P5 short primer (for Parallel-Spli
  • Linear amplification was performed as follows: 72 °C for 5 min, 98 °C for 30 sec, then 98 °C for 10 sec, 59 °C for 30 sec, and 72 °C for 1 min, for 12 cycles. Then stored at 15 °C until use.
  • RNA-seq libraries were amplified using SI-PCR Primer B (PN-2000128, 10x Genomics) and N7xx primers.
  • RNA-seq libraries were amplified using SI-PCR Primer B (PN-2000128) and P3xx primers. After amplification, ATAC-seq parts were cleaned up using 1.2x SPRI beads and RNA-seq parts were cleaned up using 0.8x SPRI beads.
  • PCR amplification mix 25 ⁇ l NEBNext High-Fidelity 2X PCR Master Mix (M0541L, NEB), 2.5 ⁇ l N5xx primer, 1.25 ⁇ l P5xx primer, 1.25 ⁇ l P3xx primer and 15 ⁇ l UltraPure DNase/RNase-Free distilled water
  • the cycling conditions were 72 °C for 5 min, 98 °C for 30 sec, then 98 °C for 10 sec, 65 °C for 30 sec, 72 °C for 1 min for 5 cycles, hold at 4 °C.
  • the PCR mix was divided into ATAC-seq part and RNA-seq part.
  • RNA-seq part was cleaned up using 1.0x AMPure XP beads (A63881, Beckman Coulter) and 0.8x AMPure XP beads, respectively.
  • the PCR products were eluted with 22 ⁇ l UltraPure DNase/RNase-Free distilled water.
  • the second round of PCR amplification was performed by adding 28 ⁇ l PCR reaction mixture (25 ⁇ l NEBNext High-Fidelity 2X PCR Master Mix, 1.25 ⁇ l N5xx primer, 1.25 ⁇ l P3_end primer, 0.5 ⁇ l 25x SYBR Green I (S7563, ThermoFisher) for ATAC-seq; 25 ⁇ l NEBNext High-Fidelity 2X PCR Master Mix, 1.25 ⁇ l N5xx primer, 1.25 ⁇ l P3_end primer, 0.5 ⁇ l 25x SYBR Green I for RNA-seq).
  • Parallel-Seq libraries were sequenced using the Illumina NovaSeq 6000 sequencing system with 16nt i5 index, 8nt i7 index, and PE150 sequencing.
  • Parallel-Split-Seq libraries were sequenced using the Illumina HiSeq X 10 System or NovaSeq 6000 Sequencing System with standard PE150 sequencing with 8nt i5 index and 8nt i7 index.
  • RNA-seq library starts at the second strand synthesis annealing site, which is identical to the RNA sequence of the target gene.
  • Raw reads were trimmed with cutadapt. Barcodes were parsed by FREE Difference software, allowing only one edit per round of barcoding. Data with embedded end sequences were filtered out from RNA libraries, and data without embedded end sequences were filtered out from ATAC libraries. Data were aligned to hg38, mm10, or the combined genome using STAR.
  • RNA-seq For single-cell RNA-seq, a modified python script from the Split-seq pipeline was used to collapse UMIs and generate digital gene expression matrices. For single-cell ATAC-seq, mitochondrial reads were removed. Enrichment of TSS accessibility was calculated as previously described to assess data quality. Cells with TSS enrichment ⁇ 6 were discarded. Tn5 insertions were then calculated on 2-kb bins across the genome.
  • X...X and “N...N” representing bases in the sequence of the present application can represent any natural or modified base type or base type known in the prior art, wherein “X” and “N” can be used interchangeably, including but not limited to A, T, C, G or U.
  • V represents A, C or G.
  • B represents C, G, T or U.
  • X represents an amino acid
  • it represents a natural or modified amino acid type known in the prior art.
  • transposon-specific barcode sequence Tn5ME-B is shown in SEQ ID NO: 1
  • sequence Tn5ME-x (x represents 1-27) with a first index is shown in SEQ ID NO: 2
  • XXXXXX in the sequence represents the first index
  • Tn5ME-B GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 1)
  • Tn5ME-x /5Phos/TGCAGTA XXXXXX AGATGTGTATAAGAGACAG (SEQ ID NO: 2)
  • R1BxT15VN /5Phos/ TGCAGTAXXXXXXTTTTTTTTTTTTTTTTTVN (SEQ ID NO: 3).
  • R1BxN6 /5Phos/TGCAGTA XXXXXX NNNNNN (SEQ ID NO: 4)
  • dscB′ sequence TACTGCACTCAGTGACT (SEQ ID NO: 5)
  • the second PCR anchor is attached to the cDNA, wherein the primer used for the second strand synthesis is the p3 short primer.
  • p3 short primer CAGACGTGTGCTCTTCCGATCTNNNGGNNNB (SEQ ID NO: 7)
  • Lysing cells adding droplet-specific marker p5 adapter, i.e., the third index, for linear amplification in the droplets, as shown in Table 2, wherein the linear amplification primer is shown in (SEQ ID NO: 8), wherein XXXXXXXXXXXXXXXXX is the third index information, which is the specific index of beads in each droplet;
  • the purified product in each PCR tube was divided into two parts, and the transcriptome and the open chromatin fragment were amplified using the corresponding primers, wherein the transcriptome was amplified using primers SI-PCR primer B (SEQ ID NO: 9) and P3xx primer (SEQ ID NO: 10), and XXXXXXX in the sequence represents the fourth index of the primer sequence required for amplifying the transcriptome, see P3xx index in Table 2; the open chromatin fragment was amplified using primers SI-PCR primer B (SEQ ID NO: 9) and N7xx primer (SEQ ID NO: 11), and XXXXXXXX in the sequence represents the fourth index of the primer sequence required for amplifying the open chromatin fragment, see N7xx index in Table 1;
  • SI-PCR Primer B AATGATACGGCGACCACCGAGA (SEQ ID NO: 9)
  • P3xx primer CAAGCAGAAGACGGCATACGAGAT XXXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 10)
  • N7xx primer CAAGCAGAAGACGGCATACGAGAT XXXXXXX GTCTCGTGGGCTCGG (SEQ ID NO: 11)
  • Parallel Seq The data quality of Parallel-Seq was further compared with sci-CAR, paired-Seq, SNARE-Seq, and SHARE-Seq.
  • Parallel Seq showed better data quality than the state-of-the-art method SHARE Seq on two libraries ( Figures 10-11), with more ATAC fragments and RNA UMIs, more captured genes, and greater bandwidth than other methods.
  • Parallel-Split-Seq was developed, which changed the location of the third index in Parallel-Seq, that is, the third index was added from linear amplification in the droplet to adding a round of ligation reaction on the plate. It still includes the step of linear amplification in the droplet, but the third index is not added in this step, and the barcode space is 24x96x96x96 ⁇ 2.12x10 7 ( Figure 12).
  • Parallel-Split-Seq (same steps as in Example 1) was performed using a mixture of NIH/3T3 (mouse), HEK293T (human), Hela (human), K562 (human) and THP1 (human) cells.
  • the specific steps are as follows:
  • Tn5ME-A TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO:12)
  • each well contains an R2′ sequence (SEQ ID NO: 13) when the second index is added, and each well contains an R3′ sequence (SEQ ID NO: 14) when the third index is added, and ligating the well-specific adapter sequence to the transposed chromatin or the first chain of cDNA, wherein the well-specific adapter sequence with the second index is shown as R2Bx (SEQ ID NO: 15), and XXXXXXX in the sequence represents the second index, as shown in Table 2; the well-specific adapter sequence with the third index is shown as R3Bx (SEQ ID NO: 16), and XXXXXXX in the sequence represents the third index, as shown in Table 2;
  • R2′ sequence TACTGCAGCTGAACCTC (SEQ ID NO: 13)
  • R3′ sequence TCTCCAAAGCTGTGGAC (SEQ ID NO: 14)
  • R2Bx sequence /5Phos/TTGGAGA XXXXXXX GAGGTTCAGC (SEQ ID NO: 15)
  • R3Bx sequence CAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNN XXXXXXX GTCCACAGCT (SEQ ID NO: 16).
  • Second-strand synthesis with random primers The second PCR anchor point is attached to the cDNA, wherein the primer used for the second-strand synthesis is the p5 short primer.
  • P5 short primer ACACGACGCTCTTCCGATCTNNNGGNNNB (SEQ ID NO: 17)
  • Each PCR product was purified and divided into two parts.
  • the transcriptome and accessible chromatin fragments were amplified using corresponding primers, wherein the transcriptome was amplified using primers p3 end (SEQ ID NO: 20) and P5xx (SEQ ID NO: 18), and XXXXXXX in the P5xx sequence was the fourth index of the amplified transcriptome, as shown in Table 3;
  • the open chromatin fragment was amplified using primers p3 end (SEQ ID NO: 20) and N5xx (SEQ ID NO: 19), and XXXXXXXX in the N5xx sequence was the fourth index of the amplified open chromatin fragment, as shown in Table 3.
  • N5xx sequence AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TCGTCGGCAGCGTC (SEQ ID NO: 19)

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Abstract

La présente invention concerne un procédé de construction d'une banque de séquençage de cellules individuelles. Le séquençage est effectué sur une banque de séquençage de cellules individuelles construite pour réaliser une analyse par paire de l'accessibilité de l'ARN et de la chromatine dans une même cellule individuelle.
PCT/CN2023/098377 2023-06-05 2023-06-05 Procédé de construction d'une banque de séquençage de cellules individuelles Ceased WO2024250155A1 (fr)

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WO2021189679A1 (fr) * 2020-03-27 2021-09-30 中国人民解放军陆军军医大学 Méthode de construction d'une banque de séquençage de transcriptome de cellule unique et son utilisation
US20220259586A1 (en) * 2017-05-26 2022-08-18 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US20220356461A1 (en) * 2019-12-19 2022-11-10 Illumina, Inc. High-throughput single-cell libraries and methods of making and of using
CN115478098A (zh) * 2022-10-10 2022-12-16 中国科学技术大学 一种单细胞转录组及染色质可及性双组学测序文库构建方法及测序方法
CN115537408A (zh) * 2022-10-08 2022-12-30 厦门大学 一种单细胞多组学文库及其构建方法
CN116949132A (zh) * 2023-06-05 2023-10-27 清华大学 一种构建单细胞测序文库的方法

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CN109996892A (zh) * 2016-12-07 2019-07-09 深圳华大智造科技有限公司 单细胞测序文库的构建方法及其应用
EP3553180A1 (fr) * 2016-12-07 2019-10-16 MGI Tech Co., Ltd. Méthode de construction d'une banque de séquençage de cellule unique et son utilisation
US20220259586A1 (en) * 2017-05-26 2022-08-18 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US20220356461A1 (en) * 2019-12-19 2022-11-10 Illumina, Inc. High-throughput single-cell libraries and methods of making and of using
WO2021189679A1 (fr) * 2020-03-27 2021-09-30 中国人民解放军陆军军医大学 Méthode de construction d'une banque de séquençage de transcriptome de cellule unique et son utilisation
CN115537408A (zh) * 2022-10-08 2022-12-30 厦门大学 一种单细胞多组学文库及其构建方法
CN115478098A (zh) * 2022-10-10 2022-12-16 中国科学技术大学 一种单细胞转录组及染色质可及性双组学测序文库构建方法及测序方法
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