WO2024250155A1 - Method for constructing single cell sequencing library - Google Patents

Abstract

Provided is a method for constructing a single cell sequencing library. Sequencing is performed on a constructed single cell sequencing library to achieve pairwise analysis of RNA and chromatin accessibility in a same single cell.

Description

A method for constructing a single-cell sequencing library Technical Field

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.

Background Art

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.

Among multimodal single-cell sequencing technologies, sci-CAR, SNARE-Seq, Paired-Seq, SHARE-Seq, and 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. However, sci-CAR barcodes have low binding and high collision rates, and 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.

In the droplet-based single-cell sequencing (DSC-seq) method, two cells will receive the same barcode when loaded into the same droplet, called a doublet, which affects single-cell data analysis. DSC-seq loads far fewer cells than the droplets it produces to avoid doublets. For example, the 10x Genomics Chromium platform produces about 100k droplets containing barcoded beads and barcoded reagents, but can only recover 10k single cells at a collision rate of about 10%. More than 80% of functional droplets never receive single cells, wasting most of the reagents and leading to high costs for large-scale studies.

Therefore, 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).

Summary of the invention

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. Compared with previous multimodal single-cell analysis methods, Parallel-Seq only performs four rounds of barcode indexing through one round of ligation reaction and two rounds of amplification reaction, and 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. Parallel-Seq and Parallel-Split-Seq were benchmarked with several human and mouse cell lines and applied to primary cells of human lung cancer samples. The results showed that the library had good data specificity, high quality, and a large number of captured genes. Moreover, there were very few doublets and the collision rate was extremely low. The method for constructing a single-cell sequencing library in the present application has a super-label combination space that can perform large-scale cell atlas projects at a lower cost.

In a first aspect of the present invention, a method for constructing a single-cell sequencing library is provided, the method 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.

Preferably, 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.

Preferably, the method further comprises synthesizing a second strand of cDNA.

Preferably, 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.

Preferably, the method further comprises purifying DNA and amplifying cDNA and chromatin DNA of the transcriptome library using primers, respectively.

Preferably, the method further comprises adding RNase.

Preferably, the method further comprises obtaining cells, fixing and permeabilizing the cells.

In a specific embodiment of the present invention, a method for constructing a single-cell sequencing library comprises:

a) using a transposon to cut the open chromatin to obtain a DNA fragment carrying a first linker;

b) adding a reverse transcription primer to reverse transcribe the mRNA to obtain the first strand of cDNA carrying the second linker;

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;

d) synthesizing the second strand of cDNA;

e) Use primers to amplify cDNA and chromatin DNA of the transcriptome library respectively.

The steps a) and b) can be performed simultaneously or sequentially. For example, step a) can be performed first and then step b), or step b) can be performed first and then step a).

Preferably, step a) is performed first and then step b).

Preferably, 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. In a specific embodiment of the present invention, 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.

Preferably, 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.

In a specific embodiment of the present invention, the reverse transcription primer comprises poly(T) and sequences complementary to the first index and subsequent primers.

In a specific embodiment of the present invention, the first adapter and the second adapter may contain the same sequence complementary to the subsequent primer.

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, 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.

Preferably, 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. CATTGAGC, CCAAGCGT, CCACGACT, CATTGTC, CCGCATGT, CCTACTCC, CCTCCTTG, CCTTAATG, CGAATATC, CGAGAGCA, CGCCTCAA, CGCGTTAC, CG GACTCT, CGGTTGTT, CGTAGCTT, CGTGCCAA, CTACCGGA, CTAGCAGT, CTCAGCCT, CTCTTCTA, CTGCTGGT, CTGTATTC, CTTCGCTC, GAAGAGTA, GACAC CTA, GACGTGAG, GACTTACT, GAGGACAA, GAGTTAAG, GATCCTCG, GCAATCCG, GCAGTGTG, GCCGCTAA, GCGACCAT, GCTAAGAC, GCTGTAGG, GGAACTGG, At least one of GGACAGTT, GGATTGCT, GGTCCTAA, GTACCTGT, GTCAAGGA, GTCTGCTT, GTGCTCCA, GTGTGACC, GTTATTGG, TAATTCGG, TACCAATC, TAGACTCC, TAGTCAAC, TCACGTTG, TCAGAATG, TCCAGCTT, TCCTGCGA, TCGGTTCC, TCTTACCT, TGACATGG, TGCCTATA, TGGTGTGG, and TGTACTAG, or a combination of two or more.

Preferably, the first vector-specific adapter comprises a second index, a UMI, and a sequence complementary to a reverse transcription primer or a transposon sequence.

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, the first vector-specific linker comprises SEQ ID NO: 6.

In a specific embodiment of the present invention, the vector contains SEQ ID NO: 5.

In another specific embodiment of the present invention, the first vector-specific linker comprises SEQ ID NO: 15.

In another specific embodiment of the present invention, 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.

Preferably, the primers used in the amplification reaction in the droplet include a third index.

In one specific embodiment of the present invention, the primer used for the amplification reaction in the droplet comprises SEQ ID NO: 8.

Preferably, the step of lysing the droplets is further included after the linear amplification. In a specific embodiment of the present invention, the lysed droplets are lysed by using a demulsifier.

Preferably, 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. Preferably, the second vector-specific adapter comprises a third index.

In a specific embodiment of the present invention, the second vector-specific linker comprises SEQ ID NO: 16.

In a specific embodiment of the present invention, the vector contains SEQ ID NO: 14.

In a specific embodiment of the present invention, 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, CGCCAGTA, CGCGGAAT, CGGAA GGA, CGGTGAGA, CGTAACAC, CGTCTATG, CGTTCTCG, CTACTAAG, CTAGTGCG, CTCTGACA, CTGATGAA, CTGGTACA, CTTACGAG, GAACTCAA, GAATGTTG, G ACGAATT, GACTGCCA, GAGCTATT, GAGTCGGA, GATAGAAC, GATGGTCT, GCAGCACT, GCATTCAT, GCCTCTGT, GCGCAGAT, GCTCACAA, GCTTGCGT, GTAATG At least one, or a combination of two or more of CA, GTATCGAG, GTCGATCT, GTGAGCGT, GTGGATAG, GTTAGCCA, TAAGGTGG, TACACCGG, TACTCGTC, TAGCTGAG, TCAACAGG, TCACTCAC, TCATAGAC, TCCGTACA, TCGGAGTA, TCGTCGGT, TGAACGCG, TGAGTCTT, TGCGACTG, TGGTTATC, TGTGTAAG, TTAGGAAC, TTCAGTGG, and TTCTATCC.

Preferably, 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.

Preferably, the fourth index comprises a combination of at least one, two or more than three of the P3xx indexes;

Preferably, the fourth index includes a combination of at least one, two or more than three of N7xx;

Preferably, the fourth index comprises a combination of at least one, two or more than three of P5xx;

Preferably, the fourth index includes a combination of at least one, two or more than three of N5xx.

To add a fourth index (e.g., P3xx index), the primers used to amplify the transcriptome are SEQ ID NO: 9, 10.

To add the fourth index (e.g. P5xx), the primers used to amplify the transcriptome are SEQ ID NO: 20, 18.

To add the fourth index (e.g. N7xx), the primers used to amplify the open chromatin fragments are SEQ ID NO: 9, 11.

To add the fourth index (e.g. N5xx), the primers used to amplify the open chromatin fragments are SEQ ID NO: 20, 19.

In a specific embodiment of the present invention, the carrier comprises a well, a tube or a plate.

Preferably, the carrier is an ELISA plate such as a 96-well plate.

Preferably, 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.

Preferably, the method further comprises obtaining cells, fixing and permeabilizing the cells.

In a second aspect of the present invention, a method for constructing a multi-mode single-cell sequencing library is provided, wherein the construction method 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.

Preferably, 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.

Preferably, the first vector-specific linker comprises a second index.

Preferably, the method further comprises the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets.

Preferably, the formed droplets are overloaded with cells;

Preferably, the primers used in the amplification reaction in the droplet include a third index.

Preferably, 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.

Preferably, the primers used in the amplification reaction performed after the DNA purification comprise a fourth index.

Preferably, 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.

Preferably, 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.

Preferably, the first vector-specific linker comprises a second index.

Preferably, the method further comprises the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets.

Preferably, the formed droplets are overloaded with cells;

Preferably, the primers used in the amplification reaction in the droplet include a third index.

Preferably, 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.

Preferably, 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.

In a sixth aspect, 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.

Preferably, the indexes are one, two, three, four, five, six, seven, eight, nine or more than ten.

Preferably, the index includes a first index, a second index, a third index and/or a fourth index.

In a specific embodiment of the present invention, 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.

Preferably, 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.

The "chromatin accessibility" of the present invention 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.

The "droplets" of the present invention are oil-in-water or water-in-oil structures. Different droplets may have different identifiers. Preferably, the aqueous mixture is combined with an oil phase. Preferably, 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. For example, "overloaded cells in a droplet" means exceeding the amount of cells carried in the original droplet. In the prior art, droplet-carrying cells include empty cells, cells carrying a single cell, or overloaded cells. Among them, 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.

The "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.

"Complementarity" as used herein refers to nucleotide sequences that are related by the base pairing rules. For example, the 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.

The "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.

As used herein, "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 "and/or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the question. For example, "A and/or B" includes "A", "A and B" and "B". For another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, 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. At the same time, 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. By design, 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. In addition, we applied Parallel-seq to lung cancer samples and demonstrated its ability to identify cis-regulatory elements in accessible regions of specific genes. Joint analysis of gene expression and chromatin accessibility was applied in tumor samples, and joint analysis and newly developed analysis methods were used to identify possible regulatory elements, including enhancers and mutations of oncogenes. In addition, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

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 4: Enrichment of scATAC-seq reads around TSSs.

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.

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.

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, and 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.

Figure 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.

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, and 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.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Cell culture method in the embodiment:

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.

Lung cancer sample preparation in the embodiment:

Fresh NSCLC solid tumor tissues were collected from PLA General Hospital and placed in precooled MACS tissue storage solution (130-100-008, Miltenyi Biotec) (2-8°C). The samples had to be fully covered with MACS and transported from the hospital to the laboratory.

A separation mixture of 4677 μL DMEM/F-12 (11320033, ThermoFisher), 250 μL 2.5 mg/ml Liberase TL (05401020001, Sigma Aldrich) to a final concentration of 250 μg/mL, 23 μl 2 mg/mL elastase (NC9301601, Worthington) to a final concentration of 9.2 μg/mL, and 50 μL 10 mg/mL DNase (11284932001, Sigma Aldrich) to a final concentration of 100 μg/mL was used.

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 μm 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 μL fetal bovine serum. 5 μL of cells were taken, mixed with 5 μL 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. Cells were resuspended with 80μL cell staining buffer (420201, BioLegend) and 5μL Human TruStain FcX (422302, BioLegend) and incubated at 4°C for 5 min. 5μL anti-CD45 PE (304039, BioLegend), 5μL anti-CD3 BV421 (317344, BioLegend) and 5μL anti-EpCam PE/Cy7 (324222, BioLegend) antibodies were added respectively and incubated at 4°C in the dark for 15 min. Wash the stained cells with 1 mL PBS and centrifuge at 500 g for 5 min at 4°C. Discard the supernatant and resuspend the single cells with 1 mL PBS containing 0.02 μM Calcein-AM (425201, BioLegend) and incubate at room temperature for 15 min in the dark. Resuspend the cells with 90 μL Annexin V Binding Buffer, 5 μL APC Annexin V (640941, BioLegend) and 5 μL 7-AAD Viability Staining Solution (420404, BioLegend), respectively. Incubate at room temperature for 10 min. Add 400 μL PBS and filter the cells with a 35 μm BD cell filter (352235, BD Falcon). 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.

The method for preparing the transposon in the embodiment is as follows:

Tn5Merev (/5Phos/CTGTCTCTTATACACATCT (SEQ ID NO: 21)), Tn5ME-A, Tn5ME-B and barcoded R1BxME (x represents 1-96) were prepared. 10μM Tn5Merev, 10μM Tn5ME-A (for Parallel-Split-Seq) or 10μM Tn5ME-B (for Parallel-Seq) were annealed with 10μM R1BxME at 95℃ for 2 minutes and gradually dropped to 20℃ and 4℃ at 0.1℃/s. Combine 2 μL annealed Tn5ME-B (Parallel-Seq), 2 μL annealed Tn5ME-R1Bx, 2 μL 10x TPS, 4 μL transposase (M0221, Robustnique), and 10 μL UltraPure DNase/RNase-Free Water (10977023, ThermoFisher) and incubate at room temperature for 30 min. Aliquot 4 μL of the assembled transposon into each tube and store at -20°C for no more than 1 month.

The method for fixing cells in the embodiment is as follows:

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 250μL PBS. Add 750μL PBS containing 1.33% methanol-free formaldehyde (28906, ThermoFisher) and incubate in ice for 10 minutes. Add 50μL 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.

The method for permeabilizing cells in the embodiment is as follows:

2x RSB was prepared as Omni ATAC by mixing 1 mL 1M Tris HCl pH 7.4 (T2663-1L, Sigma-Aldrich), 200 μL 5M NaCl (AM9759, ThermoFisher), 300 μL 1M MgCl ₂ (AM9530G, ThermoFisher), and 48.5 mL ultrapure DNase/RNase free distilled water. During fixation, prepare permeabilization buffer, combine 50 μL 2x RSB, 1 μL RiboLock (EO0384, ThermoFisher), 1 μL SUPERase·In RNase inhibitor (AM2696, ThermoFisher), 1 μL 10% Nonidet P40 substitute (1133247301, Sigma-Aldrich), 1 μL 10% tween20 (11332465001, Sigma-Aldrich), 1 μL 1% Digitonin (D141-100MG, Sigma-Aldrich), 5 μL 20% BSA, 40 μL ultrapure DNase/RNase free distilled water per sample. After removing the fixative reagent, immediately add 100 μL of permeabilization buffer, pipette 8 times, and incubate in ice for 5 minutes. After permeabilization, add 1 mL of wash buffer to each sample. Centrifuge the cells and remove the supernatant.

The method of transposition in the embodiment is as follows:

Prepare ATAC-seq reaction solution by mixing 10 μL 5xLM buffer (M0221, Robustnique), 16.5 μL PBS, 0.5 μL RiboLock, 0.5 μL SUPERase·In RNase Inhibitor, 0.5 μL 10% Tween 20, 0.5 μL Digiton.5 μL, and 17.5 μL ultrapure DNase/RNase free distilled water. Resuspend permeabilized single cells with 46 μL ATAC-seq reaction solution and add 4 μL barcode-specific transposon to each tube. ATAC-seq reactions were performed at 37°C and 550 r.p.m. with a heated lid. After the ATAC-seq reaction, 949 μL PBS, 10 μL 10% Triton X-100 (93443, Sigma-Aldrich), 1 μL RiboLock, and 50 μL 20% BSA were added to each tube and centrifuged to remove the supernatant.

The method of reverse transcription in cells in the embodiment is as follows:

Prepare 16 μL of resuspension solution for each sample by mixing 8 μL PBS, 0.5 μL RiboLock, 0.5 μL SUPERase·In RNase Inhibitor, and 7 μL nuclease-free water. Prepare reverse transcription mixture by adding 8 μL 5x RT buffer, 2 μL 10 mM dNTPs (N0447L, NEB), 0.5 μL RiboLock, 0.25 μL SUPERase·In RNase Inhibitor, 4 μL Maxima H Minus Reverse Transcriptase (EP0753 and ThermoFisher), and 5.25 μL UltraPure DNase/RNase-Free distilled water. Split 20 μL of reverse transcription mixture into each tube and add 2 μL of barcode-matched random and 2 μL of polyT reverse transcription primers). Resuspend transposed cells with 16 μL of resuspension solution and add to barcode-matched PCR tubes. Mix well, reverse transcribe at 50°C for 10 minutes, then go through 3 thermal cycles (8°C for 12 seconds, 15°C for 45 seconds, 20°C for 45 seconds, 30°C for 30 seconds, 42°C for 120 seconds, and 50°C for 180 seconds), incubate at 50°C for 5 minutes, and store permanently at 4°C. Combine the reverse transcription reactions into a 1.5mL tube on ice. Centrifuge the cells and remove the supernatant. Wash the cells again with 1mL PBS supplemented with 10μL 10% Triton X-100 and 50μL 20% BSA. Centrifuge the cells and remove the supernatant.

The connection reaction method in the embodiment is as follows:

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. Prior to intracellular barcode ligation, the ligation adapter was annealed by combining 11μM of the ligation strand and 12μM of the barcode strand in a 100μL 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 10μL of ligation adapter.

For Parallel-Split-Seq, 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.

Among them, 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 μL 10x T4 DNA ligation buffer, 100 μL T4 DNA ligase (M0082, Robustnique), 50 μL 10% Triton x-100 and 1350 μL 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 μL 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. After ligation, add 2 μL 500 μM EDTA (AM9260G, ThermoFisher) to each well and combine. The combined cells were added with 50 μl 10% Triton X-100, 50 μL 20% BSA and centrifuged to remove the supernatant. The cells were washed again with 940 μL PBS, 10 μL 10% Triton X-100, and 50 μL 20% BSA. For Parallel-Split-Seq, the third index was added by ligation reaction after the second index tagging.

The method of RNase digestion in the embodiment is as follows:

Resuspend cells using RNase digestion reaction (40 μL 5xRT buffer, 8 μL RNase Cocktail Enzyme Mix (AM2286, ThermoFisher), 8 μL RNAse H (Y9220L, Enzymatics) and 144 μL 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 μL PBS and 10 μL 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.

The method for second chain synthesis in the embodiment is as follows:

Incubate the cells with the second-strand synthesis reaction mixture (40 μL 5xRT buffer, 48 μL 50% PEG 8000 (B1004SVIAL, NEB), 20 μL 10 mM dNTPs, 2 μL 1 mM dN-P3 short primer (for Parallel-Seq) or dN-P5 short primer (for Parallel-Split-Seq), 5 μL Klenow Exo- (M0212L, NEB) and 85 μL 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. After second-strand synthesis, wash the cells twice with PBS containing 0.1 Triton X-100 and 1% BSA. Then resuspend the cells with 40 μL 0.5xPBS and count them in trypan blue using a C-Chip disposable hemocytometer.

Overload with 10x Chromium ATAC-seq kit

For Parallel-Seq, cells were centrifuged and resuspended with 7 μL ATAC-seq Buffer B and made up to 15 μL with 1x Nucleies Buffer. 56.5 μL Barcoding Reagent B, 1.5 μL Reducing Agent B, and 2 μL Barcoding Enzyme (PN-1000176, 10x Genomics) were combined with cells and loaded into one channel of Chromium Next GEM Chip H (PN-1000162, 10x Genomics). After GEM generation, droplets were divided into 16 tubes, each containing 6.25 μL droplets. 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.

Parallel-Seq library construction

With Chromium Next GEM Single Cell ATAC Reagent Kits V1.1 scaled down, perform post-GEM incubation cleanup. Add 7.8 μL Recovery Agent to each tube and gently invert the tube 10 times to mix. Centrifuge briefly and add 12.5 μL Dynabeads Cleanup Mix. Pipette mix 5 times and incubate at room temperature for 10 min. Elute product with 81 μL Elution Solution I and split into two parts, 40 μL for ATAC-seq and 40 μL for RNA-seq. Clean up the ATAC-seq part using 1.2x SPRI beads (B23318, Beckman Coulter) and the RNA-seq part using 0.8x SPRI beads. Amplify ATAC-seq libraries 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.

Parallel-Split-Seq library construction

Dilute the second chain synthesis cells to 800 cells/μl and split 2,000 cells per tube. Add 2.5 μl 2x lysis buffer (0.25 μl 1 M pH 8.0 Tris-HCl, 0.25 μl 10% IGEPAL CA-630 (I8896, Sigma Aldrich), 0.25 μl 10% Tween 20, 0.5 μl 291 mg/ml QIAGEN Protease (19155, QIAGEN) and 1.25 μl UltraPure DNase/RNase-Free distilled water) and incubate at 55 °C for 8 h, incubate at 70 °C for 15 min to inactivate QIAGEN Protease and keep permanently at 4 °C. 45 μl PCR amplification mix (25 μl NEBNext High-Fidelity 2X PCR Master Mix (M0541L, NEB), 2.5 μl N5xx primer, 1.25 μl P5xx primer, 1.25 μl P3xx primer and 15 μl UltraPure DNase/RNase-Free distilled water) was added to amplify ATAC-seq and RNA-seq fragments. 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. The 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 μl UltraPure DNase/RNase-Free distilled water. The second round of PCR amplification was performed by adding 28 μl PCR reaction mixture (25 μl NEBNext High-Fidelity 2X PCR Master Mix, 1.25 μl N5xx primer, 1.25 μl P3_end primer, 0.5 μl 25x SYBR Green I (S7563, ThermoFisher) for ATAC-seq; 25 μl NEBNext High-Fidelity 2X PCR Master Mix, 1.25 μl N5xx primer, 1.25 μl P3_end primer, 0.5 μl 25x SYBR Green I for RNA-seq). We amplified each sublibrary of ATAC-seq and RNA-seq libraries on a QuantStudio 3 Real-Time PCR System (ThermoFisher), followed the amplification and stopped each sublibrary when the fluorescence unit value reached ∼100,000. As a rule of thumb, the cycle number is about 6–8 for cell lines or tumor cells and about 7–10 for other primary cells, with less than 11 being acceptable. ATAC-seq libraries were cleaned up using 1.0x AMPure XP beads and RNA-seq libraries were cleaned up using 0.8x AMPure XP beads, respectively. Libraries were eluted with 20 μl of elution buffer (19086, QIAGEN) and the concentration was determined using the Qubit dsDNA HS Assay Kit (Q32851, ThermoFisher). More than 20 ng of product was expected to be recovered for each sublibrary. Quality control was performed using the Agilent High Sensitivity D1000 ScreenTape Assay (5067-5584 and 5067-5585, Agilent).

The sequencing method in the embodiment is as follows:

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.

Preprocessing of Parallel-Seq and Parallel-Split-Seq Data

We use read1 to sequence the cell barcodes and adapters for Parallel-Seq. To balance the sequencing nucleotide composition, we add phase nucleotides between barcode2 and the adapter, no nucleotides are added at positions 1-24, T is added at positions 25-48, CA is added at positions 49-72, and ACA is added at positions 73-96. For barcode2's ^1-24th , Parallel-Seq's barcode1, barcode2, barcode3, and barcode4 should be located within 36-41st, 11-18th, i5 index, i7 index. For barcode2's ^25-48th , ^49-72nd , and ^73-96th positions, only the position of barcode1 needs to be changed by one nucleotide step. The unique molecular identifier is located within read1's ^1-10th . The sequence following the Tn5ME in read1 is a Tn5 cleavage site, while read2 provides another Tn5 cleavage site for ATAC-seq. Read2 of the RNA-seq library starts at the second strand synthesis annealing site, which is identical to the RNA sequence of the target gene.

For Parallel-Split-Seq, use read2 to sequence the cell barcodes and ligation adapters. barcode1, barcode2, barcode3, and barcode4 should be located within indexes 61-66, 36-43, 11-18, and i7. The sequence following Tn5ME in read2 is a Tn5 cut site, while read1 provides another fragment for ATAC-seq. Read1 of the RNA-seq library provides 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.

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.

Cells expressing <200 genes and <200 bins were discarded. We used Scrublet to predict doublet probabilities and removed doublets using the default threshold.

For mixed cell line data, cells with <90% UMI mapped to one species were considered mixed cells.

The control method in the embodiment is as follows:

For the steps of sci-CAR, please refer to the literature: Cao, J. et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science 361, 1380-1385 (2018).

For the steps of paired-Seq, please refer to the literature: Zhu, C. et al. An ultra high-throughput method for single-cell joint analysis of open chromatin and transcriptome. Nat Struct Mol Biol 26, 1063-1070 (2019).

For the steps of SNARE-Seq, please refer to the literature: Chen, S., Lake, B.B. & Zhang, K. High-throughput sequencing of the transcriptome and chromatin accessibility in the same cell. Nat Biotechnol 37, 1452-1457 (2019).

For the steps of SHARE-Seq, please refer to the literature: Ma, S. et al. Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin. Cell 183, 1103-1116 e20 (2020).

The "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. Of course, when "X" represents an amino acid, it represents a natural or modified amino acid type known in the prior art.

Example 1 Parallel-Seq analysis of RNA and open chromatin in the same single cell from multiple samples

The experimental design of Parallel-Seq is shown in Figure 1. The specific steps are as follows:

(1) Parallel-Seq started with 27 different samples, each with 50,000 single cells;

(2) The cells of each sample were fixed, permeabilized and used with a barcoded Tn5 transposon, and the open chromatin was labeled with a transposon-specific barcode; wherein the transposon-specific barcode sequence Tn5ME-B is shown in SEQ ID NO: 1, and the sequence Tn5ME-x (x represents 1-27) with a first index is shown in SEQ ID NO: 2, and XXXXXX in the sequence represents the first index, see Table 1.

Tn5ME-B: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 1)

Tn5ME-x:/5Phos/TGCAGTA XXXXXX AGATGTGTATAAGAGACAG (SEQ ID NO: 2)

(3) Reverse transcription of mRNA of each sample was performed using barcode-matched poly(T) primer R1BxT15VN and random hexamer primer R1BxN6. The reverse transcription primers R1BxT15VN and R1BxN6 (x represents 1-27) are shown in SEQ ID NO: 3 and SEQ ID NO: 4. XXXXXX in the sequence represents the first index, see Table 1.

R1BxT15VN:/5Phos/ TGCAGTAXXXXXXTTTTTTTTTTTTTTTVN (SEQ ID NO: 3).

R1BxN6:/5Phos/TGCAGTA XXXXXX NNNNNN (SEQ ID NO: 4)

(4) Cells of different samples were combined and randomly distributed into a 96-well plate, each well containing a dscB′ sequence (SEQ ID NO: 5), and the well-specific adapter sequence dscBx was connected to the transposed chromatin or the first strand of cDNA, wherein the well-specific adapter sequence dscBx (x represents 1-96) is as shown in SEQ ID NO: 6, "NNNNNNNNNN" in the sequence is the UMI, a phase nucleotide is added between the second index and the UMI, no nucleotides are added to dscB1-dscB24, T is added to dscB25-dscB48, CA is added to dscB49-dscB72, and ACA is added to dscB73-dscB96, and XXXXXXXX in the sequence represents the second index, see Table 2;

dscB′ sequence: TACTGCACTCAGTGACT (SEQ ID NO: 5)

dscBx sequence: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNN XXXXXXXX AGTCACTGAG (SEQ ID NO: 6)

(5) digesting RNA with RNase;

(6) Random Primer Second Strand Synthesis: 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)

(7) All cells are pooled together and overloaded into one channel of the Chromium scATAC-seq chip;

(8) 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 XXXXXXXXXXXXXXXX is the third index information, which is the specific index of beads in each droplet;

5'-AATGATACGGCGACCACCGAGATCTACAC- XXXXXXXXXXXXXXXX -TCGTCGGCAGCGTC-3'(SEQ ID NO: 8)

(9) further dividing the droplets into 16 PCR tubes for PCR purification;

(10) 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 XXXXXXXX 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 XXXXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 10)

N7xx primer: CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGG (SEQ ID NO: 11)

(11) After sequencing and barcode parsing analysis, the gene expression and chromatin accessibility profiles of the same combination of four rounds of indexing represent the pairwise profiles of a single cell. In principle, after four rounds of indexing, the barcode space is largely expanded to (96x96x100000x16≈1.47x10 ¹⁰ ), which enables Parallel-Seq to evaluate more than 1 million cells in a single experiment with a very low collision rate.

Table 1

Table 2

Example 2 Parallel-Seq performance verification

Parallel-Seq was performed with a mixture of NIH/3T3 (mouse), HEK293T (human), and K562 (human) cells (same steps as in Example 1), and the transcriptome and chromatin accessibility of 2200 cells were obtained after quality screening, of which the average UMI of the scRNA sequence part was 7014, and the average UMI of the scATAC sequence part was 10103. Reads from human and mouse cells were well separated in both transcriptome and chromatin profiles, with transcriptomes assigned to 802 mouse cells and 1398 human cells, and chromatin profiles assigned to 805 mouse cells and 1398 human cells, with very few doublets, and the collision rates of the two maps were 0.2% and 0.1%, respectively (Figure 2).

The insert size distribution of the aggregated scATAC-seq data showed a clear nucleosome binding pattern (Figure 3), and the TSS enrichment score of the sequencing reads was as high as 14 (Figure 4), indicating that the scATAC-seq data was qualified.

The aggregated single-cell chromatin accessibility and transcriptome profiles generated by Parallel-Seq were closely correlated with the bulk DNA sequence (ENCFF156LGK, R=0.79) (Figure 5) and nuclear RNA sequence (ENCFF631TDY, R=0.81) (Figure 6) of K562 cells in ENCODE (Figure 7). In addition, the expression and chromatin accessibility profiles of each cell clustered together within the cell type and separated from each other (Figures 8-9). Together, these data demonstrate the high specificity and high quality of 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.

Example 3 Parallel-Split-Seq and performance verification

In order to make it easier to use and further reduce costs, 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).

In this example, 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:

(1) Fixing and permeabilizing the cells of each sample and using a barcode Tn5 transposon to label the open chromatin with a transposon-specific barcode; wherein the transposon-specific barcode sequence Tn5ME-A is shown in SEQ ID NO: 12, and the sequence Tn5ME-x (x represents 1-27) with a first index is shown in SEQ ID NO: 2, and XXXXXX in the sequence represents the first index, as shown in Table 1;

Tn5ME-A：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO：12)

(2) The mRNA of each sample was reverse transcribed using the barcode-matched poly(T) primer R1BxT15VN and the random hexamer primer R1BxN6. The reverse transcription primers R1BxT15VN and R1BxN6 (x represents 1-27) are shown in SEQ ID NO: 3 and SEQ ID NO: 4. XXXXXX in the sequence represents the first index, see Table 1.

(3) combining cells of different samples and randomly distributing them into a 96-well plate, performing two ligation reactions, adding a second index and a third index, wherein 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 XXXXXXXX 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 XXXXXXXX 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 XXXXXXXX GAGGTTCAGC (SEQ ID NO: 15)

R3Bx sequence: CAGACGTGTGCTCTTCCGATCTNNNNNNNNNN XXXXXXXX GTCCACAGCT (SEQ ID NO: 16).

(4) digesting RNA with RNase;

(5) 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)

(6) All cells were pooled together, counted and diluted to 800 cells/ul, and dispensed into PCR tubes, with 2.5ul cells per tube;

(7) Lyse the cells in a PCR tube and directly add the PCR amplification system (including P5xx (SEQ ID NO: 18), N5xx (SEQ ID NO: 19) and P3xx (SEQ ID NO: 10)) for amplification, and add the fourth index, see Table 2;

(8) 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 XXXXXXXX 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.

p3 end sequence: CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 20)

P5xx sequence:

AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX ACACTCTTTCCCTACACGACCGCTTCCGATCT (SEQ ID NO: 18)

N5xx sequence: AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TCGTCGGCAGCGTC (SEQ ID NO: 19)

Table 3

The results show that Parallel-Split-Seq has good specificity, low collision rate, and high correlation with large amounts of data (see Figures 13-18). Moreover, the performance of Parallel-Split-Seq is comparable to that of Parallel-Seq and is superior to existing methods (see Figures 19-20).

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims (18)

A method for constructing a single-cell sequencing library, comprising: a) using a transposon to cut the open chromatin to obtain a DNA fragment carrying a first linker; b) adding a reverse transcription primer to reverse transcribe the mRNA to obtain the first strand of cDNA carrying the second adapter; 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; d) synthesizing the second strand of cDNA; e) and using primers to amplify cDNA and chromatin DNA of the transcriptome library, respectively. The method according to claim 1, characterized in that the transposon comprises a barcode sequence and a transposase; Preferably, the barcode sequence comprises a first adapter; Preferably, the barcode sequence comprises a first index. The method according to claim 1 or 2 is characterized in that the reverse transcription primer comprises a second linker, and the second linker comprises poly (T) and a first index; preferably, it also comprises a random hexamer primer. The method according to any one of claims 1 to 3 is characterized in that 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 of them. The method according to any one of claims 1 to 4, characterized in that the first vector-specific linker comprises a second index, preferably, 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, ATATCACG, ATCGCCGT, ATGACGGA, ATGGAATG, ATTCCTAC, CAACGCCA, CAAGTCTG, CACACATC, CACCTTAT, CAGAACCT , CAGCCGAT, CATACTGT, CATCCACC, CATTGAGC, CCAAGCGT, CCACGACT, CCATTGTC, CCGCATGT, CCTACTCC, CCTCCTTG, CCTTAATG, CGAATATC, CGAGAGCA , CGCCTCAA, CGCGTTAC, CGGACTCT, CGGTTGTT, CGTAGCTT, CGTGCCAA, CTACCGGA, CTAGCAGT, CTCAGCCT, CTCTTCTA, CTGCTGGT, CTGTATTC, CTTCGCTC ,GAAGAGTA,GACACCTA,GACGTGAG,GACTTACT,GAGGACAA,GAGTTAAG,GATCCTCG,GCAATCCG,GCAGTGTG,GCCGCTAA,GCGACCAT,GCTAAGAC,GCTGTAGG , GGAACTGG, GGACAGTT, GGATTGCT, GGTCCTAA, GTACCTGT, GTCAAGGA, GTCTGCTT, GTGCTCCA, GTGTGACC, GTTATTGG, TAATTCGG, TACCAATC, TAGACTCC, TAGTCAAC, TCACGTTG, TCAGAATG, TCCAGCTT, TCCTGCGA, TCGGTTCC, TCTTACCT, TGACATGG, TGCCTATA, TGGTGTGG, and TGTACTAG. The method according to any one of claims 1-5, characterized in that the method further comprises the steps of forming droplets, lysing cells and performing an amplification reaction in the droplets, preferably, the formed droplets are overloaded with cells. The method according to claim 6, characterized in that the primers used in the amplification reaction in the droplet include a third index. The method according to any one of claims 1 to 5, characterized in that the method comprises using a second vector-specific linker to connect the DNA fragment carrying the first linker obtained in claim 1 and the first chain of the cDNA obtained in claim 1 to a vector, respectively, preferably, the second vector-specific linker comprises a third index. The method according to claim 7 or 8 is characterized in that 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, AGGTGTCT, AGTCCGTT, AGTTCGCA, ATAAGCTC, ATCCATG A.ATCTAGCG,ATGCAACC,ATGTGCAG,ATTGGTAG,CAAGAAGA,CAATGGAC,CACATGCT,CACGGTAG,CAGAGGTT,CAGTATAG,CATCAAGT,CATGTTCC,CCAACAAT,CCAATTAC,CCAGTGAA , CCGATCAG, CCGGTCTT, CGACAACG, CGCCAGTA, CGCGGAAT, CGGAAGGA, CGGTGAGA, CGTAACAC, CGTCTATG, CGTTCTCG, CTACTAAG, CTAGTGCG, CTCTTGACA, CTGATGAA, CTGGTACA, At least one of CTTACGAG, GAACTCAA, GAATGTTG, GACGAATT, GACTGCCA, GAGCTATT, GAGTCGGA, GATAGAAC, GATGGTCT, GCAGCACT, GCATTCAT, GCCTCTGT, GCGCAGAT, GCTCACAA, GCTTGCGT, GTAATGCA, GTATCGAG, GTCGATCT, GTGAGCGT, GTGGATAG, GTTAGCCA, TAAGGTGG, TACACCGG, TACTCGTC, TAGCTGAG, TCAACAGG, TCACTCAC, TCATAGAC, TCCGTACA, TCGGAGTA, TCGTCGGT, TGAACGCG, TGAGTCTT, TGCGACTG, TGGTTATC, TGTGTAAG, TTAGGAAC, TTCAGTGG, and TTCTATCC, or a combination of two or more of them. The method according to any one of claims 1 to 9, characterized in that the primers in the amplification reaction performed in step e) comprise a fourth index; Preferably, the fourth index comprises a combination of at least one, two or more than three of the P3xx indexes; Preferably, the fourth index includes a combination of at least one, two or more than three of the N7xx indexes; Preferably, the fourth index comprises a combination of at least one, two or more than three of the P5xx indexes; Preferably, the fourth index includes a combination of at least one, two or more than three of the N5xx indexes; The P3xx index, N7xx index, P5xx index or N5xx index is as follows:



The method according to any one of claims 2-10 is characterized in that the carrier comprises a hole, a tube or a plate. The method according to any one of claims 1-11, characterized in that the method further comprises adding RNase. The method according to any one of claims 1-12, characterized in that the method further comprises obtaining cells, fixing the cells and permeabilizing them. A method for constructing a transcriptome library, characterized in that the method 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 a second chain of cDNA; and purifying and amplifying the cDNA of the transcriptome using primers. A method for constructing a chromatin DNA library, characterized in that the method comprises using a transposon to cut open chromatin to obtain a DNA fragment carrying a first linker; placing a cell on a vector, and connecting the obtained DNA fragment carrying the first linker to the vector using a first vector-specific linker; purifying DNA and amplifying the chromatin DNA respectively using primers. A nucleic acid library obtained by the method according to any one of claims 1 to 15. A sequencing method, characterized in that the sequencing method comprises constructing the nucleic acid library according to claim 16. An application of the nucleic acid library according to claim 16, characterized in that the application includes tumor target screening, disease monitoring or pre-implantation embryo diagnosis.