A method for constructing a single-cell transcriptome and chromatin accessibility dual-omics sequencing library and a sequencing method

Abstract

The application discloses a method for constructing a single-cell transcriptome and chromatin accessibility double-omics single-cell sequencing library, which comprises the following steps: a) preparing a single-cell suspension; b) obtaining chromatin open sites; c) performing a reverse transcription reaction on the transcriptome of the cell by using a reverse transcriptase and a reverse transcription primer, so as to obtain the transcriptome information of the single cell; d) performing a template switching reaction by using a template switching oligo (TSO); e) subsequent coding; f) initial library amplification; and g) preparing a sequencing chromatin open site library and a transcriptome library. The application also discloses a method for sequencing a single-cell transcriptome and chromatin accessibility double-omics sequencing library, which comprises the steps of respectively sequencing the chromatin open site library and the transcriptome library prepared by the method.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, it relates to a method for constructing a single-cell sequencing library, and more specifically, to a method for constructing a single-cell transcriptome and chromatin accessibility dual-omics sequencing library, the prepared sequencing library, and a method for sequencing using the library. Background Technology

[0002] Single-cell transcriptome and chromatin accessibility dual-omics sequencing technology (hereinafter referred to as single-cell dual-omics sequencing technology in this disclosure) was first published in Science in 2018. Figure 2 Sci-CAR-Seq 1 Sci-CAR-Seq uses a combined indexing principle to encode individual cells; however, due to only two rounds of combined indexing, its theoretical throughput is only in the tens of thousands. Subsequently, researchers developed a flow cytometry-based cell sorting technology... Figure 3 scCAT-Seq 2 , 2019, 100-level flux) and based on microfluidics ( Figure 4 SNARE-Seq 3 In 2019, a single-cell dual-omics sequencing technology with a throughput of tens of thousands was developed. Improving throughput and quality remains a key direction for technological development. Because combinatorial indexing technology can provide extremely high-throughput sequencing, researchers have further developed a million-throughput single-cell chromatin accessibility and transcriptome dual-omics sequencing technology. Figure 5 Paired-Seq 4 ,2019; Figure 6 SHARE-Seq 5 (Chromium, 2020). Currently, 10X Genomics has released the first microfluidic-based single-cell chromatin accessibility and transcriptome dual-omics sequencing kit (Chromium, 2020). The latest technology developed before the patent application date can encode cells based on flow cytometry or microfluidics (ISSAC-Seq6, 2022).

[0003] The following is a brief overview of existing single-cell dual-omics sequencing technologies:

[0004] sci-CAR-Seq 1: First, cells are dispersed into different wells of a plate, mRNA is reverse transcribed into cDNA by reverse transcription reaction and cDNA from the same well is encoded with the first round of encoding, then transposon is used for transposition reaction to make chromatin open site from the same well to carry the first round of encoding, then all cells are mixed evenly and dispersed into new wells for lysis, a part of the lysate is used for amplification of cDNA library and cDNA is encoded with the second round of encoding, and the other part of the lysate is used for amplification of chromatin open site library and chromatin open site library is encoded with the second round of encoding. Finally, the combination of two rounds of encoding is used to determine the transcriptome library and chromatin open site library from the same cell (see sci-CAR-seq technical process schematic diagram Figure 2 ).

[0005] scCAT-Seq 2 : Cells are sorted into wells of a plate by laser, each well has a single cell. The cells in the air are lysed, the cytoplasm and nucleus are separated, and the cytoplasm is subjected to reverse transcription reaction to construct the transcriptome library, while the nucleus is subjected to transposition reaction to construct the chromatin open site library (see scCAT-Seq technical process schematic diagram Figure 3 ).

[0006] SNARE-Seq 3 : Prepare a single nucleus (single nucleus) suspension, perform Tn5 transposon transposition reaction on single nucleus to capture chromatin open site information, through microfluidic process, capture magnetic beads, single nucleus and splint primers are wrapped in a microdroplet, reaction is carried out in the microdroplet, chromatin open site sequence information is transferred to the magnetic beads, and mRNA information is captured by the primers on the magnetic beads. Amplify the library to obtain the transcriptome library and chromatin open site library for sequencing (see SNARE-Seq process schematic diagram Figure 4 ).

[0007] Paired-Seq 4 : Cells are evenly dispersed into different wells, transposition and reverse transcription reactions are used to make chromatin open site and transcriptome in the same well of the cells carry the same encoding, three round combination index method is used for encoding connection, and finally the combination of encoding is used to determine the chromatin open site and transcriptome sequence from the same cell (see Paired-Seq process schematic diagram Figure 5 ).

[0008] Shared-Seq 5: First, the cells are transposed, and then reverse transcription is performed using a reverse transcription primer containing a biotin label; using a combination indexing method, the cells are uniformly dispersed into different wells for encoding connection and repeated twice, and finally the chromatin open site information and transcriptome information from the same cell can be identified according to the combination of the encoding (see Share-Seq technical process schematic diagram Figure 6 ).

[0009] 10X Chromium single-cell multi-omics (ATAC + gene expression) kit: prepare a single cell nucleus (mononuclear) suspension, perform a Tn5 transposon transposition reaction on the mononuclear, capture chromatin open site information, and through a microfluidic process, wrap the capture magnetic beads, mononuclear and reaction system in a microdroplet, and perform the reaction in the microdroplet, while encoding the chromatin open site and transcriptome library of the cell.

[0010] ISSAC-seq 6 : Prepare a single cell nucleus (mononuclear) suspension, perform a Tn5 transposon transposition reaction on the mononuclear, capture chromatin open site information; then perform a reverse transcription reaction to capture transcriptome information, and subsequently, through a microfluidic process, wrap the capture magnetic beads, mononuclear and reaction system in a microdroplet, and perform the reaction in the microdroplet, while encoding the chromatin open site and transcriptome library of the cell; or it can also be selected to use a flow sorting method to sort into a well plate for encoding connection.

[0011] However, in all current single-cell dual-omics sequencing technologies, mainly the 3' end information of messenger RNA is obtained, and the 5' information of messenger RNA is not retained, which leads to certain limitations in the use of the current single-cell dual-omics sequencing technology. For example, the current dual-omics technology cannot conveniently sequence the commercial sgRNA sequence library for gene editing screening (CRISPR-Screen), and by obtaining the sgRNA sequence and the transcriptome and chromatin open site change information after gene editing or gene regulation, the research on the transcriptional regulation network and the cell signal transduction network can be facilitated. At the same time, 3' sequencing has certain limitations in compatibility with TCR sequencing and BCR sequencing, making it difficult to trace back through TCR and BCR information in single-cell research of the immune system. Through 5' sequencing, TCR and BCR information can be obtained, and immune cells can be traced back, which can further correct the evolution trajectory of immune cells and obtain more reliable results, which is very important for the study of immune plasticity. The technical principle demonstrated by the present disclosure can solve these problems and other related needs. SUMMARY

[0012] The inventors of this application have designed a high-throughput single-cell transcriptomics and chromatin accessibility dual-omics sequencing library construction technology capable of capturing messenger RNA 5' information. Specifically, the technical problems existing in this field are solved through the technical solutions shown in the following projects.

[0013] 1. A method for constructing a single-cell transcript and chromatin accessibility dual-omics single-cell sequencing library, comprising the following steps:

[0014] a) Prepare a single-cell suspension;

[0015] b) Obtaining chromatin open sites, which includes performing a transposition reaction on the chromatin of the cells using a Tn5 transposon assembled with primer dimers and Tn5 transposase, cleaving the chromatin open sites of the cells, and ligating primer dimers to the chromatin open sites;

[0016] c) Use reverse transcriptase and reverse transcription primers to perform reverse transcription on the transcriptome of cells to obtain transcriptome information of single cells;

[0017] d) Using template-switching oligomers (TSO) for template switching reactions;

[0018] e) Subsequent encoding to obtain a transcriptome library and a chromatin open site library with cell coding, wherein the subsequent encoding includes single-cell isolation or coding integration of cells using different platforms or processes as needed, wherein the transcriptome library and the chromatin open site library from the same cell have the same cell coding or combination of cell coding.

[0019] f) Initial library expansion, which includes expanding the previously constructed library to increase the number of usable library fragments;

[0020] g) Preparation of chromatin open site libraries and transcriptome libraries for sequencing, including the isolation of chromatin open site libraries and transcriptome libraries from the libraries amplified in the previous step.

[0021] The template conversion oligomer ( Figure 1 (As shown) includes a sequence ⑤ that is fully or partially complementary to the sequence ⑧ generated by the terminal transferase activity of reverse transcriptase, such that the reverse transcriptase can continue sequence synthesis using the template-converting oligomer as a template. The template-converting oligomer also includes a partial structural region ④ located at the 5' end of the sequence ⑤ and a handle sequence ③ located at the 5' end of the partial structural region ④ for subsequent coding integration. Optionally, the template-converting oligomer also includes a sequence ⑥ that is complementary to the partial structural region ④ to form a double-stranded structure with the template-converting oligomer.

[0022] 2. The method described in Project 1, wherein the single-cell suspension in step a) is used without or with a fixative, such as formaldehyde, for subsequent steps.

[0023] 3. The method of item 2, wherein the fixation is followed by a post-fixation that is either terminated or not terminated for subsequent steps.

[0024] 4. The method of any one of items 1 to 3, wherein the primer dimer, the template switch oligo, the reverse transcription primer is modified, for example, by phosphorylation modification and biotin modification.

[0025] 5. The method of any one of items 1 to 4, wherein the Figure 1 sequence (vi) is 3’ phosphorylated and can be dephosphorylated in subsequent reactions, for example, by a polynucleotide kinase reaction.

[0026] 6. The method of any one of items 1 to 5, wherein the template switch oligo is attached to a medium, such as a bead, a magnetized bead, a microwell, a chip, a plate, or is in a free state.

[0027] 7. The method of any one of items 1 to 6, wherein the Figure 1 the partial structure region (iv) comprises one or more functional regions, which can be selected from, but not limited to, unique molecular identifier (UMI), partial coding and double-stranded complementary sequences, etc.

[0028] 8. The method of any one of items 1 to 7, wherein the Figure 1 sequence (v) consists of DNA, RNA, locked nucleic acid, or a combination thereof.

[0029] 9. The method of any one of items 1 to 4, wherein the Figure 1 sequence (vi) is 5’ phosphorylated.

[0030] 10. The method of any one of items 1 to 9, wherein the initial library amplification is performed by a PCR reaction, the primers used comprise a coding structure, as part of a cellular coding combination, and / or comprise a coding of sample origin, experimental batch, or cell type.

[0031] 11. The method of any one of items 1 to 10, further comprising further amplification and purification of the isolated chromatin open site library and the transcriptome library.

[0032] 12. The method of any one of items 1 to 11, further comprising adding sequencing universal adapters to the isolated chromatin open site library and / or adding sequencing universal adapters to the isolated transcriptome library.

[0033] 13. The method of any one of items 1 to 12, wherein the transcriptome comprises, but is not limited to, mRNA encoding T cell receptor (TCR), B cell receptor (BCR), and / or guide RNA in CRISPR system, and the transcriptome library comprises a TCR library, a BCR library, and / or a gRNA library.

[0034] 14. A single-cell transcriptome and chromatin accessibility dual-ome sequencing library prepared by the method of item 1 to 13.

[0035] The present technology is named as USTC-V-Seq herein, which adopts a novel barcode linkage mode, can link barcodes to chromatin open sites or / and transcriptomes and retain 5' information of transcriptomes, and can combine chromatin open sequence fragments and transcriptome sequences from the same cell with the same barcode combination. Meanwhile, due to the presence of special sequences, gRNA library and BCR or / and TCR library sequences can be further enriched from the obtained transcriptome library, and corresponding gRNA sequencing, BCR sequencing and TCR sequencing can be completed. Based on this, USTC-V-Seq can simultaneously obtain chromatin accessibility information and transcriptome information from the same cell and can be compatible with gRNA information (if contained), BCR sequence information (if contained) and TCR sequence information (if contained).

[0036] The design of template switching in the present application is original. In the general knowledge background, most people would think that the newly synthesized complementary strand would fill in the template switching strand in the template switching reaction, and the flowchart or schematic diagram of other technologies would also default to fill in. In theory, the barcode cannot be linked to the template switching oligomer. But this consensus is not entirely correct. In our experiments, we found that even if the template switching oligomer is single-stranded, the barcode can be linked to the template switching strand after the transposition reaction, and it has been verified by multiple experiments. The underlying mechanism has not been verified, but it is speculated that the template switching oligomer is not completely reacted, so the barcode integration handle site is connected with the barcode, thereby realizing the barcode of the cell. Adding double-stranded is also to further block the progress of the template switching reaction. In actual situations, the barcode can be integrated into the barcode integration handle site of the single-stranded template switching oligomer or the double-stranded template switching oligomer. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 . Schematic diagram of obtaining transcriptome.

[0038] Figure 2 . Schematic diagram of sci-CAR-seq technical process.

[0039] Figure 3 . Schematic diagram of scCAT-Seq technical process.

[0040] Figure 4 . Schematic diagram of SNARE-Seq process.

[0041] Figure 5Paired-Seq workflow schematic.

[0042] Figure 6 Share-Seq workflow schematic.

[0043] Figure 7 Integration of subsequent encoding schematic.

[0044] Figure 8 Cell encoding effect diagram.

[0045] Figure 9 Transcriptome data gene alignment diagram.

[0046] Figure 10 Chromatin open site and transcriptome data correlation. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific examples and with reference to the drawings. Unless specifically stated otherwise in this text, the terms in this application have the meanings commonly understood by those skilled in the art.

[0048] Definitions

[0049] Encoding (barcode): The encoding (barcode, barcoding or index) or encoding combination described herein refers to different base sequences composed of nucleic acids, for example ATCG and TACG are two different encodings.

[0050] Cell: The basic component element of mammalian (such as human, mouse) life activities. The cell in the present disclosure is not limited to the whole cell, but also refers to other cell components, such as nucleus, mitochondria, etc. For example, a single cell suspension can also be a single nucleus suspension.

[0051] Chromatin: A linear complex structure composed of DNA, histone, non-histone and a small amount of RNA in the nucleus. The basic unit is the nucleosome formed by the winding of DNA on histone.

[0052] Chromatin accessibility: It refers to the evaluation of whether a segment of DNA is wound on histone. Generally, there are two cases of chromatin: 1) DNA is tightly wound on the nucleosome, which is called closed DNA; 2) DNA is wound on the nucleosome and is in a naked state, which is called open DNA.

[0053] Chromatin accessibility library: A sequencing library composed of sequences of chromatin open sites.

[0054] Chromatin Accessibility Sequencing (ATAC-seq): A sequencing technology developed by Stanford University in 2012 to detect the chromatin accessibility of biological samples (>500 cells).

[0055] Genome: The complete DNA sequence of an organism, composed of the four bases A, B, C, and D arranged in an orderly manner. The genomes of major mammals such as humans and mice have been fully sequenced.

[0056] Gene: A gene (hereditary factor) is the complete DNA sequence required to produce a polypeptide chain or functional RNA. A gene is generally one or more segments of DNA in the genome.

[0057] Transcriptome: Also known as the transcriptome, broadly it refers to the entirety of all RNA that a cell can transcribe, including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and non-coding RNA; narrowly it refers to all messenger RNA (mRNA) that a cell can transcribe. This disclosure uses a specific definition, namely all RNA that meets the library construction requirements, including but not limited to mRNA and gRNA in the CRISPR system.

[0058] Antigen: refers to a substance that can induce antibody production; it is any substance that can trigger an immune response.

[0059] Antibody: A protective protein produced by the body in response to antigen stimulation.

[0060] B cell receptor (BCR): A molecule located on the surface of B cells that is responsible for specifically recognizing and binding antigens; it is essentially an immunoglobulin on the cell membrane.

[0061] T cell receptor (TCR): A specific receptor located on the surface of T cells, responsible for recognizing antigens presented by the major histocompatibility complex (MHC); however, unlike B cell receptors, it cannot recognize free antigens.

[0062] B-cell receptor / T-cell receptor sequencing (BCR-Seq / TCR-Seq): a sequencing technology targeting the sequences of B-cell receptors / T-cell receptors.

[0063] Transcription factors: Proteins that recognize specific DNA sequence patterns (motifs) and bind to DNA, and can initiate or regulate gene expression.

[0064] Single-cell chromatin accessibility sequencing (scATAC-seq): A sequencing method used to detect the accessibility of chromatin in a single cell.

[0065] Single-cell transcriptome sequencing (scRNA-Seq): A sequencing method used to detect the transcriptome of a single cell.

[0066] Single-cell multi-omics sequencing: Sequencing library construction methods used to detect multiple dimensions of information in a single cell, such as sequencing library construction methods that simultaneously obtain dual-omics or more omics information from the same cell, such as transcriptome, chromatin accessibility and / or proteome information (e.g., sci-CAR, Paired-Seq, Shared-Seq, 10X Chromium, CITE-Seq, REAP-Seq, etc.).

[0067] The "template-switching oligomers (TSOs)" described in this article can be named by other names or be unnamed. Essentially, they are single-stranded or double-stranded nucleic acid sequences that can be attached to media including, but not limited to, beads, magnetized beads, micropores, chips, and plates, or can exist in a free state. Template-switching oligomers (TSOs) may contain functional regions including, but not limited to, encoding unique molecular identifiers (UMIs) and subsequent encoding integration handle sites.

[0068] The “template switching reaction” mentioned in this article generally refers to the reaction in which, under the action of reverse transcriptase, a template switching oligomer is used as a template to continue sequence synthesis, generating a sequence that is complementary or partially complementary to the template switching oligomer.

[0069] Preparation of single-cell suspension

[0070] Non-free tissue and / or free tissue (such as blood) can be prepared into single-cell suspensions through various methods, including but not limited to cutting, grinding, and enzymatic hydrolysis. The tissue can be in a healthy state or in a diseased state, and the tissue's state includes, but is not limited to, fresh tissue, frozen tissue, and tissue sections. In some embodiments, it may be necessary to extract cell nuclei; the extraction process includes, but is not limited to, grinding, permeabilization treatment, and flow cytometry sorting.

[0071] Fixation of single-cell suspension

[0072] In some embodiments, the single-cell suspension may be used without fixation or in subsequent processes. In other embodiments, the cell concentration in the single-cell suspension needs to be determined and a suitable fixation system (containing a fixative) is used to achieve the desired fixation effect. Suitable fixatives include, but are not limited to, aldehydes such as formaldehyde, glutaraldehyde, and paraformaldehyde, alcohols such as methanol and ethanol, and acetone, and can be used at any suitable level or concentration.

[0073] In some embodiments, fixation can be terminated after fixation, using methods including but not limited to any suitable level or concentration of glycine and bovine serum albumin, or other reagents or methods. In some embodiments, subsequent reactions can proceed directly without terminating fixation.

[0074] Acquisition of chromatin open sites

[0075] The designed primer dimer can be incubated with Tn5 transposase to assemble a Tn5 transposon that can be used for transposition. The appropriate Tn5 transposition system is prepared, and the Tn5 transposon is used to perform the transposition reaction, cleaving the chromatin opening sites of the cell and ligating the primer dimer to the chromatin opening sites. The primer dimer usually contains a handle sequence encoding subsequent integration.

[0076] Methods for designing primer dimers are well known in the art. In some embodiments, primer dimers can be designed or modified as needed. This document only provides one example of an implementable scheme and does not limit the sequences used in this invention.

[0077] Tn5 transposase can be synthesized or purified in-house, or purchased from a supplier, including but not limited to VAZYME S601-01. The primer-transposase incubation system can be prepared in-house or commercially available. The incubation and transposition reaction conditions can be adjusted.

[0078] In some implementations, any component of the primer dimer may be modified, including but not limited to phosphorylation and biotin modification.

[0079] Acquisition of transcriptome information

[0080] Reverse transcription can be performed on cells using reverse transcription primers. After the reverse transcription reaction is complete, the cells can be incubated at 42°C for a period of time, and then template conversion oligomers (TSO) can be used for template conversion. The template conversion oligomers can provide the reverse transcription product with a handle sequence that encodes subsequent integration.

[0081] The result of the template switching reaction is the integration of template-switched oligomers, complementary products of template-switched oligomers, or partially complementary products of template-switched oligomers into the library sequence.

[0082] In some embodiments, the "template conversion reaction" can be replaced by, but is not limited to, reverse transcription, ligation, and DNA polymerization reactions. In some embodiments, the reverse transcriptase can be, but is not limited to, mouse leukemia virus (MMLV)-based reverse transcriptases and their derivatives. In some embodiments, the reverse transcription primers can be modified, including but not limited to biotin modification.

[0083] The incubation time at 42°C can be adjusted as needed. Incubation at 42°C can also be omitted. Generally, incubation at 42°C can improve yield to some extent.

[0084] Template conversion oligomers include, but are not limited to, modified or unmodified double-stranded template conversion oligomers or modified or unmodified single-stranded template conversion oligomers. For example, to prevent double-stranded TSO from elongating under the action of reverse transcriptase, which would lead to the closure of the coding linkage site, while ensuring the ligation efficiency of double-stranded TSO, double-stranded template conversion oligomers can be modified by, but are not limited to, 3' phosphorylation modification and locked nucleic acid modification.

[0085] In some implementations, double-stranded template conversion oligomers (TSOs) can be formed by annealing two nucleic acid sequences.

[0086] In some embodiments, the modifications referred to as modified double-stranded template-conversion oligomers (TSOs) include, but are not limited to, containing only or simultaneously 5' phosphorylation, 3' phosphorylation, and locked nucleic acids.

[0087] In some embodiments, TSO modification needs to be removed; after template conversion, an enzyme capable of removing one or more specific modifications is used to remove them, exposing the double-stranded TSO to chemical groups that can proceed with subsequent reactions. In some embodiments, the modification may not affect the subsequent reaction steps described herein, so removal of the specific modification is not necessarily required.

[0088] In some embodiments, the special modifications described herein may be modified by 3' phosphorylation. In this case, it may or may not be necessary to use a reaction including but not limited to T4 polynucleotide kinase (NEBM0201S or NEB M0201L) to remove the special modifications.

[0089] In the transcriptome information acquisition procedure, a handle site was provided on the template-converting oligomer to facilitate subsequent coding integration, thus ensuring that the coding was integrated at the 5' end of the mRNA sequence. Other single-cell dual-omics sequencing technologies have not adopted this coding integration method. Simultaneously, an incubation step at 42°C was introduced to increase product yield.

[0090] existFigure 1 The diagram illustrates transcriptome acquisition. This step allows for the acquisition of a portion of the 5' coding sequence and subsequent coding sequences. Figure 1 ① shows a single chain in template conversion oligomer (TSO) single or double chain, which includes three parts: ③, ④ and ⑤. Figure 1 Figure ② shows the transcriptome, including but not limited to mRNA and gRNA in the CRISPR system. Figure 1 Figure ③ shows the subsequent encoding and integration of the handle sequence. The sequence length is a range and can be adjusted according to the experiment. In some implementations, this segment can also be removed. Figure 1 Figure ④ shows a partial structural region of TSO. This part may include one or more functional regions, including but not limited to unique molecular recognition signals (UMI), partial coding (functions include but are not limited to cell recognition, tissue recognition, etc.), double-stranded complementary sequences, etc. Figure 1 As shown in ⑤, Figure 1 The complementary bases in group ⑧, and the number of M, is a range that can be adjusted according to experiments. M can be the same base or different bases, and the specific bases can be determined based on... Figure 1 The N in ⑧ is used to determine the M; M can be, but is not limited to, deoxyribonucleic acid, ribonucleic acid, and locked nucleic acid. Figure 1 As shown in ⑥, it is related to Figure 1 A complementary sequence in region ④ can form a double-chain structure with the template-transformed oligomer to increase the likelihood that region ③ is a single chain; furthermore... Figure 1 The sequence shown in ⑥ can be modified, including but not limited to 3' phosphorylation, 5' phosphorylation, etc. Figure 1 ⑦ is based on Figure 1 Part ④ of the sequence is a newly generated sequence from the template. Figure 1 In the middle, ⑧ represents the sequence generated under the terminal transferase activity of reverse transcriptase. The length is a range, and the specific sequence is also related to the reverse transcriptase used. Figure 1 The sequence shown in Figure 9 is the one synthesized using reverse transcription primers.

[0091] Subsequent encoding

[0092] Different platforms or processes can be used to isolate or encode cells as needed. These platforms or processes include, but are not limited to, cell coding technologies based on microfluidics, flow cytometry, and combined indexing. Figure 7 The diagram illustrates the integration step of subsequent coding. Through this step, subsequent coding can be integrated into the 5' end of the corresponding RNA in the transcriptome library, simultaneously ensuring that transcriptome libraries and chromatin open site libraries from the same cell carry the same coding. Figure 7Figure A shows the library structure before subsequent coding steps, where ① shows the library structure of open chromatin sites after transposition, where X represents the open genome sequence; ② shows the transcriptome library structure after template conversion, where M and N have the following meanings: Figure 1 ⑤ and ⑧ are described in the diagram; ③ is a simplified version of ① and ②. The coding integration principles of transcriptome libraries and chromatin open site libraries are similar, so the following diagram uses the schematic diagram of ③ to replace chromatin open site libraries and transcriptome libraries; ④ shows the coding integration handle site; ⑤ shows the other end protruding region, which can be designed as needed and can be the same as or different from ④, and can be used for coding integration or other purposes. Figure 7 B shows a schematic diagram of a media capture library (applicable to, but not limited to, microfluidic technology, micropore technology, etc.), where ⑥ is a structural sequence complementary to ⑦. The complex formed by ⑥ and ⑦ can be integrated with the coding integration handle site. At least one of ⑥ and ⑦ contains a sequence including but not limited to cell coding information. At least one of ⑥ and ⑦ is combined with the media interface ⑧. ⑧ is the media interface, which may include, but is not limited to, a plane, a curved surface, a sphere, etc. Figure 7 C shows the integration of the coding into library ③ based on the amplification method (applicable to technologies including but not limited to flow cytometry sorting). ④ is the coding integration handle site, which may not be used in the amplification method. Instead, sequences including but not limited to cell coding information are added to primer ⑨ to integrate the coding into library ③. ⑩ is the amplification primer at the other end, which may or may not contain sequences including but not limited to coding information as needed. Figure 7 D shows a coding integration method mediated by non-media and amplification methods (applicable to, but not limited to, combined indexing techniques, streaming sorting techniques, etc.), and ④ is the coding integration handle site. and This forms the first round of coding sequence, which can be integrated with site ④. and This forms the second round of coding sequence, which can be combined with... The free ends are integrated, and a third or higher round of coding can be designed as needed. In some implementations, one or more rounds of coding can be used for integration.

[0093] The term "integration" or "combination" includes, but is not limited to, integration or combination based on complete sequence complementarity, integration or combination based on partial sequence complementarity, or connection integration or combination. The results of such integration or combination include, but are not limited to, the formation of a double-chain structure or a single-chain structure.

[0094] The aforementioned preparation of a reaction system from cells and beads using microfluidic technology can utilize any suitable cells or cell components (such as cell nuclei).

[0095] The “subsequent reactions” include, but are not limited to, ligation reactions and polymerase chain amplification (PCR) reactions.

[0096] In some implementations, cells can be sorted into reaction units using sorting techniques. Coding integration handles on chromatin open sites and transcriptomes can bind to or react with substances in the reaction unit to integrate new codes into the chromatin open sites and transcriptome sequences.

[0097] The sorting technology mentioned includes, but is not limited to, flow sorting technology.

[0098] The reaction unit includes, but is not limited to, the pores of a perforated plate or the pores of a microplate.

[0099] The “binding or reaction” mentioned includes, but is not limited to, binding based on sequence complementarity, binding based on partial sequence complementarity, or binding by linkage.

[0100] The term "binding or reaction" includes, but is not limited to, ligation reactions and polymerase chain amplification (PCR) reactions.

[0101] The results of the "binding or reaction" include, but are not limited to, the formation of a double-chain structure or a single-chain structure.

[0102] The “encoding” mentioned includes, but is not limited to, originating from a single chain or a double chain.

[0103] In some implementations, a handle location may not be included.

[0104] In some implementations, cells can perform multiple rounds of coding based on a combinatorial index. After homogenization, cells are randomly dispersed into a set containing several reaction units. Handle sites on chromatin open site sequences and TSOs can bind to or react with substances in the coding reaction units of the combinatorial index for coding integration. After each round of integration, unintegrated coding is blocked using a designed blocker to prevent interference with subsequent processes. If there are multiple rounds of combinatorial indexing, after each round of integration, the chromatin open site sequences and transcriptome sequences are integrated into handle sites available for subsequent coding integration. Through one or more rounds of coding integration, new coding can be integrated into the chromatin open site sequences and transcriptome sequences.

[0105] The reaction unit includes, but is not limited to, the pores of a perforated plate or the pores of a microplate.

[0106] The term "collection of several reaction units" includes, but is not limited to, orifice plates or connecting pipes.

[0107] The “subsequent reactions” include, but are not limited to, ligation reactions and polymerase chain amplification (PCR) reactions.

[0108] In some implementations, cell lysis is required. Cells are mixed thoroughly and then distributed into separate wells (≥1) for lysis, with each well containing ≥0 cells. This releases chromatin open site sequences and transcriptome sequences for subsequent processes.

[0109] The pyrolysis reaction may need to be terminated under certain circumstances to avoid interference with subsequent processes.

[0110] In some implementations, nucleic acid sequences attached to a medium can be simultaneously integrated into open sites on chromatin and the transcriptome for encoding, and the encoding integration step can be skipped to proceed to subsequent processes.

[0111] This invention utilizes handle sites on template-converting oligomers to integrate subsequent coding onto the template-converting oligomers, thereby integrating the coding at the 5' end of the mRNA sequence. Alternatively, the coding can be designed onto amplification primers to add coding at the 5' end relative to the mRNA. In contrast, other known single-cell dual-omics technologies in the art integrate coding at the 3' end of the mRNA sequence.

[0112] Initial library amplification

[0113] The constructed library needs to be expanded to increase the number of usable library fragments.

[0114] In some implementations, the cell lysate containing the encoded amplification primers and other polymerase chain reaction (PCR) systems can be added to the terminated cell lysate, and the PCR reaction can be performed.

[0115] The primers may contain coding structures and may be used as part of a cell coding combination, or may be adjusted as needed to include, but not limited to, coding for sample source, experimental batch, cell type, etc.

[0116] In some implementations, beads that integrate library information can be placed into a library amplification system to perform library amplification.

[0117] Encoding cells by adding codes to amplification primers has been reported in the art. However, the encoding of the present invention can not only encode cells, but also encode and mark sample source, experimental batch, cell type, etc., as needed, so as to distinguish them when integrating data from multiple samples, batches, and cell types in the future.

[0118] Preparation of sequencing chromatin open site libraries and transcriptome libraries

[0119] In some implementations, streptavidin magnetic beads can be used to bind and capture biotin-tagged chromatin open site libraries or transcriptome libraries, and the mixture of magnetic beads and libraries can be placed on a magnetic rack for separation; biotin-free transcriptome libraries or chromatin open site libraries will remain in the supernatant, while biotin-tagged chromatin open site libraries or transcriptome libraries will bind to the magnetic beads and be adsorbed by the magnetic rack.

[0120] In some implementations, the separated chromatin open site libraries and transcriptome libraries may be further amplified and purified as needed. The purification includes, but is not limited to, removal of impurities and fragment length screening.

[0121] In some implementations, the isolated chromatin open site library may lack sequencing adapters and cannot be sequenced, so it is necessary to add sequencing universal adapters to the library. The reactions for adding sequencing universal adapters include, but are not limited to, PCR reactions and ligation reactions.

[0122] In some implementations, amplification and purification are required after adding the universal sequencing adapter. This purification includes, but is not limited to, impurity removal and fragment length screening.

[0123] In some implementations, the isolated full-length transcriptome library may lack sequencing adapters and cannot be sequenced. The purified transcriptome library is then processed to add sequencing adapters. This "processing" includes, but is not limited to, single or multiple steps such as Tn5 transposition, nuclease digestion, PCR reaction, and ligation reaction.

[0124] Preparation of special sequence libraries

[0125] If the obtained transcriptome library contains special sequence libraries such as T cell receptor sequences, B cell receptor sequences, and gRNA sequences, it can be enriched using relevant primers if necessary (including but not limited to PCR amplification and biotin-streptavidin magnetic bead capture). If the enriched T cell receptor sequencing library, B cell receptor sequencing library, and gRNA sequence sequencing library do not have sequencing adapters, they need to be further processed to obtain universal sequencing adapters for the special sequence sequencing libraries, including but not limited to using Tn5 transposition reaction, PCR reaction, ligation reaction, etc.

[0126] Because the coding is integrated at the 5' end of the mRNA sequence, it can preserve the cell coding combination while enriching special sequence libraries such as T cell receptor sequences, B cell receptor sequences and gRNA sequences, thus enabling the preparation and sequencing of related special sequence libraries.

[0127] Example

[0128] The following examples are provided to help better understand the present invention, but are not intended to limit the invention.

[0129] Unless otherwise specified, the experimental methods in the following examples are all conventional experimental methods, and the reagents used are all readily available.

[0130] The experimental reagents, equipment, and specific experimental conditions described in the following examples have been verified to enable the implementation of this invention. These reagents, equipment, and specific experimental conditions are provided for ease of understanding and do not limit the scope of the invention. The use of alternative reagents, equipment, or conditions is within the protection scope of this invention.

[0131] Example 1

[0132] In this embodiment, a mixed cell line library was prepared based on the K562 cell line and the NIH3T3 cell line. Only chromatin open site libraries and transcriptome libraries obtained by reverse transcription of mRNA were prepared (excluding special libraries such as TCR libraries, BCR libraries, CRISPR gRNA libraries, etc.).

[0133] 1. Preparation of single-cell suspension

[0134] K562 is a suspension cell line and does not require trypsin treatment, while NIH3T3 is an adherent cell line and requires treatment with a certain concentration of trypsin to suspend it.

[0135] 2. Cell fixation

[0136] Cell fixation can be performed to complete the library construction process, or this step can be omitted. Fixation can be performed by adding 66.8 μL of 1.6% formaldehyde solution to 1 ml of single-cell suspension (suspended in phosphate-buffered saline, PBS), with a cell density of 102. 6 Cells / ml. After fixation for 5 minutes, fixation was terminated for 5 minutes with fixation stop solution (56 μL 2.5M glycine, 20 μL 1M Tris-HCl 8.0, 13.4 μL 7.5% BSA), followed by centrifugation to remove cells. Cells were washed twice with PBS-BSA-RI wash buffer and resuspended in 1XTD buffer (diluted with 4XTD buffer).

[0137] Table 1. PBS-BSA-RI washing solution system

[0138] Reagent Volume (microliters, μL) 1X PBS 987.5 10% BSA 1 0.1M DTT 10 SUPERase In 1 RNaseOUT 0.5 Total 1000

[0139] Table 2. 4XTD System

[0140] Reagent Volume (microliters, μL) 1M Tris-acetate, PH 7.8 132 5M Potassium acetate 52.8 1M Magnesium acetate 40 Dimethylformamide (DMF) 640 DEPC H2O 135.2 Total 1000

[0141] 3. Obtaining chromatin open sites

[0142] To obtain chromatin open sites, the Tn5 naked enzyme was first assembled according to the system in Table 3 and incubated at room temperature for 1 hour for later use.

[0143] Table 3. Tn5 transposable assembly system

[0144]

[0145] In the Tn5 transposon assembly system shown in Table 3, the 10X assembly buffer was provided by the supplier, and the assembly reaction was performed according to the supplier's standard operating procedure. The transposon primer dimer was a double-stranded DNA structure, and the annealing system and conditions were performed according to the supplier's standard operating procedure. The assembled Tn5 transposon was transposed for 30 minutes at 37°C and 500 rpm in the reaction system shown in Table 4. After transposition, the chromatin open site sequence will contain the subsequent coding integration handle site.

[0146] Table 4. Transposable Reaction Systems

[0147]

[0148]

[0149] 4. Acquisition of transcriptome information

[0150] After transposition, an equal volume of NIB-BSA-RI(H) washing buffer (Table 6) was added, and the cells were centrifuged at 500 x g, 4 °C for 5 min. The supernatant was removed, and the washing was repeated three times. After washing, the cell pellet was resuspended in the reverse transcription system (Table 7), and a reverse transcription reaction was performed (Table 8).

[0151] Table 5. NIB System

[0152] Reagent Volume (μL) DEPC H2O 9840 1M Tris-HCl 7.5 100 5M NaCl 20 1M MgCl2 30 5% Digitonin 10 Total 10000

[0153] Table 6. NIB-BSA-RI(H) system

[0154] Reagent Volume (μL) NIB 100 10% BSA 0.5 0.1M DTT 1 SUPERase In 1 RNaseOUT 0.5

[0155] Table 7. Reverse Transcription System

[0156]

[0157] Table 8. Thermal cycling of reverse transcription

[0158]

[0159]

[0160] After the reverse transcription reaction is complete, add an appropriate volume (e.g., 50 μL) of NIB-BSA-RI(H) washing buffer, centrifuge at 500 x g, 4 °C for 5 min, remove the supernatant, and repeat the washing process twice. After washing, resuspend the cell pellet in the template conversion system (Table 9) and perform the template conversion reaction (Table 10). If the template conversion oligomer has a double-stranded structure, annealing is required first. After the template conversion reaction is complete, add an appropriate volume (e.g., 50 μL) of NIB-BSA-RI washing buffer, centrifuge at 500 x g, 4 °C for 5 min, remove the supernatant, and repeat the washing process twice. Resuspend the cells in an appropriate volume (e.g., 1152 μL) of NIB-BSA-RI(L) (Table 13). At this point, the transcriptome in the cells has provided subsequent coding integration handle sites at the 5' end of the RNA.

[0161] Table 9. Template Conversion System

[0162]

[0163] Table 10. Template conversion reaction conditions

[0164]

[0165]

[0166] Table 11. Template conversion oligomer annealing system

[0167] Reagent Volume (μL) 100 μM Template Switching Oligo Single Strand 1 10 100 μM Template Switching Oligo Single Strand 2 10 [H2O (added after annealing)] 30

[0168] Table 12. Annealing conditions for template conversion oligomers

[0169]

[0170] Table 13. NIB-BSA-RI(L) system

[0171] Reagent Volume (μL) NIB 100 10% BSA 0.5 0.1M DTT 1 SUPERase In 0.25 RNaseOUT 0.125

[0172] 5. Subsequent coding integration

[0173] Subsequent encoding integration can be carried out in different ways. This embodiment uses a composite index method, so the following steps take a composite index as an example.

[0174] Prepare the subsequent coding integration reaction system (Table 14). Mix and disperse the cells into 96-well plates containing the first-round coding sequence, and incubate at 25°C and 300 rpm for 30 min on a shaker. After the reaction, add the first-round blocking primers and incubate again at 25°C and 300 rpm for 30 min for blocking. After blocking, combine all reaction systems in the 96-well plates, mix well, add 192 μL of T4 DNA ligase, and then mix and disperse into 96-well plates containing 10 μL of the second-round coding sequence per well. Incubate at 25°C and 300 rpm for 30 min on a shaker. After the reaction, add the second-round blocking primers and incubate again at 25°C and 300 rpm for 30 min for blocking. After blocking, combine all reaction systems in the 96-well plates, centrifuge at 500 x g at 4°C for 5 min, remove the supernatant, and wash twice with NIB-BSA-RI(L).

[0175] Table 14. Subsequent Coding Integration System (Combined Index System)

[0176]

[0177] Table 15. Molecular hybridization solution system

[0178]

[0179] Table 16. First-round coded annealing scheme (for each code)

[0180]

[0181] Table 17 Primer Annealing Buffer System

[0182] Primer Annealing Buffer Volume (μl) Final Concentration 1 M Tris 8.0 10 10 mM 5 M NaCl 10 50 mM 0.5 M EDTA 2 1 mM H2O (or DEPC H2O) 978 Total 1000

[0183] Table 18. Coded Annealing Conditions

[0184]

[0185] Table 19. First Round Coding Closed System

[0186]

[0187]

[0188] Table 20. Second-round coded annealing system (for each code)

[0189]

[0190] Table 21. Second-round coding closed system

[0191]

[0192] 6. Initial library expansion

[0193] This step can involve introducing another round of coding, or it can be skipped. This embodiment introduces one round of coding at this stage; the following is the process for introducing this new round of coding.

[0194] After washing the cells, resuspend them in 1478 μL of 10 mM Tris-HCl pH 7.5 and evenly disperse them into 96-well plates (14 μL per well, with some residue). Add 2 μL of cell lysis buffer and 0.2 μL of 20 mg / mL proteinase K to each well and mix well. Then incubate at 55 °C and 500 rpm for 15 min on a shaker. After incubation, add 4 μL of 10% Tween-20 and 0.4 μL of 100 mM PMSF to each well to terminate the incubation.

[0195] Prepare the initial library amplification system, perform 10 rounds of linear amplification, then add exponential amplification primers and perform 5 more rounds of exponential amplification (the number of amplification rounds can be adjusted as needed).

[0196] Table 22. Linear amplification system

[0197]

[0198] Table 23. Thermal Cycling of Linear Amplification

[0199]

[0200] Table 24. Exponential Amplification System

[0201]

[0202]

[0203] Table 25. Thermal Cyclic Exponential Growth

[0204]

[0205] After exponential amplification, samples from all wells were collected and mixed, then purified according to the procedures of the commercial kit MinElute PCRPurification Kit (QIAGEN Cat.No. / ID:28006), and finally eluted with 50 μL of DEPCH2O. After elution, 100 μL of (2X) SPRISelect Beads (Beckman Coulter) were added for fragment purification according to the procedures. In the purified products, the transcriptome library was biotin-labeled, while the chromatin open site library was not biotin-labeled. Chromatin open site sequences and transcriptome sequences from the same cell will contain the same cell coding sequences.

[0206] The purified product was processed using (Dynabead) TM MyOne TM Streptomycin C1 (SCI) and biotinylated streptavidin magnetic beads were bound and incubated at room temperature for 1 hour. The mixture was then placed on a magnetic rack for magnetic adsorption to separate the chromatin open site library and the transcriptome library. The magnetic beads bound the transcriptome library, and the supernatant contained the chromatin open site library. The obtained supernatant was purified using a QIAGEN MinElute PCR Purification Kit and eluted in 23 μL DEPC H2O for the construction of the final chromatin open site library (ready for sequencing). The magnetic beads were washed sequentially with 50 μL 1X BW-T (Table 27), 50 μL 1X BW, and 50 μL DEPC H2O, then resuspended in 21 μL DEPC H2O to prepare the PCR reaction system, and the transcriptome library was released using PCR.

[0207] Table 26. 1X BW System

[0208]

[0209]

[0210] Table 27. 1X BW-T System

[0211] Reagent Volume (μL) 1X BW 1000 10% Tween-20 5

[0212] Table 28. Transcriptome library release system

[0213]

[0214] Table 29. Thermal cycling of transcriptome library release

[0215]

[0216] After the transcriptome library release reaction was completed, the magnetic beads were separated from the product system using a magnetic rack to obtain the product system (supernatant), which was then amplified using PCR. After amplification, the library was purified using 0.6X SPRISelect beads, and finally eluted with an appropriate volume (e.g., 20 μL) of DEPC H2O for later use. This material can be used for the construction of the final transcriptome library and the enrichment and construction of specific sequence libraries.

[0217] If a special sequence library exists in the transcriptome library, the method of initial library amplification can be simulated by using appropriate primers to enrich and separate the mRNA reverse transcription library and the special sequence library (the special sequence library can be similar to the chromatin open site library), and the final library can be constructed for sequencing.

[0218] Table 30. Thermal cycling of transcriptome library re-expansion

[0219]

[0220] Construction of the final library of 7-1 chromatin open sites

[0221] The chromatin open site library obtained in Section 6 was used to construct the final chromatin open site library using the following system and reaction. After construction, the final chromatin open site library was purified using a 0.5X-0.9X SPRISelect beads paired-end selection method and eluted in 20 μL DEPC H2O. At this point, the chromatin open site library preparation was complete, and sequencing was ready.

[0222] Table 31. Final Library Construction System for Open Chromatin Sites

[0223]

[0224] Primer 1 for the 10μM final library in Table 31 can be designed to include encoding as needed.

[0225] Table 32. Thermal cycling of final library construction at chromatin open sites

[0226]

[0227] 7-2 Construction of the final transcriptome library (If special sequences are present, this method can be used to construct the final library after isolating the special sequence library).

[0228] The transcriptome library obtained in Section 6 was used to prepare a transcriptome cutting system and incubated at a constant temperature of 55°C for 5 minutes.

[0229] Table 33. Transcriptome cleavage system

[0230] Reagent Volume (μL) Transcriptome Library 20 ng of corresponding volume (V) 4X TD Buffer 12.5 DEPC H2O 36.5-V Tn5 (same transposition complex sequence) 1 Total 50

[0231] After cleavage, 1 μL of 10% SDS was added to the cleavage system to terminate the reaction, and the mixture was incubated at room temperature for 5 min. After incubation, the product was purified using 0.6X SPRISelect beads and eluted with 23 μL of DEPC H2O. The eluted product was used to prepare the final transcriptome library reaction system and the final transcriptome library preparation reaction was performed.

[0232] Table 34. Transcriptome Final Library Construction System

[0233]

[0234]

[0235] Primer 1 for the 10μM final library in Table 34 can be designed to include encoding as needed.

[0236] Table 35. Thermal cycling of final transcriptome library construction

[0237]

[0238] After the final transcriptome library preparation was completed, the product was purified using 0.6X SPRISelect beads and eluted with 20 μL LEPC H2O. The final transcriptome library preparation was now complete; the library contains the 5' transcriptome information and is ready for sequencing.

[0239] Experimental results

[0240] Figure 8 This is a diagram illustrating the cell coding effect obtained according to an embodiment of the present invention. In one implementation, inputting 50,000 cells yielded transcriptome data for 7,138 cells and chromatin open site data for 6,665 cells. A total of 6,378 cells can simultaneously obtain transcriptome and chromatin open site data from both omics approaches, achieving an acceptable two-cell rate (~5%).

[0241] Distribution alignment of the obtained transcriptome data showed that the obtained transcriptome sequences were mainly distributed at the 5' end of the mRNA, proving that the transcriptome data of this invention can preserve the 5' information of mRNA (taking K562 as an example) (see...). Figure 9 ).

[0242] Correlation analysis revealed that chromatin opening sites and transcriptome information of characteristic genes in cell lines showed good correlation (taking K562 as an example, MALAT1 is a normally expressed housekeeping gene, and GATA1 is a characteristic gene of K562) (see...). Figure 10 ).

[0243] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0244] References

[0245] 1Cao,J.et al.Joint profiling of chromatin accessibility and geneexpression in thousands of single cells.Science 361,1380-1385,doi:10.1126 / science.aau0730(2018).

[0246] 2Liu,L.et al.Deconvolution of single-cell multi-omics layers revealsregulatory heterogeneity.Nature Communications 10,470,doi:10.1038 / s41467-018-08205-7(2019).

[0247] 3Chen,S.,Lake,B.B.&Zhang,K.High-throughput sequencing of thetranscriptome and chromatin accessibility in the same cell.NatureBiotechnology 37,1452-1457,doi:10.1038 / s41587-019-0290-0(2019).

[0248] 4Zhu,C.et al.An ultra high-throughput method for single-cell jointanalysis of open chromatin and transcriptome.Nature Structural&MolecularBiology 26,1063-1070,doi:10.1038 / s41594-019-0323-x(2019).

[0249] 5Ma,S.et al.Chromatin potential identified by shared single cellprofiling of RNA and chromatin.bioRxiv,2020.2006.2017.156943,doi:10.1101 / 2020.06.17.156943(2020).

[0250] 6Xu,W.et al.ISSAAC-seq enables sensitive and flexible multimodalprofiling of chromatin accessibility and gene expression in singlecells.bioRxiv,2022.2001.2016.476488,doi:10.1101 / 2022.01.16.476488(2022).

Claims

1. A method for constructing a single-cell transcript and chromatin accessibility dual-omics single-cell sequencing library, comprising the following steps: a) Prepare a single-cell suspension; b) Obtaining chromatin open sites, which includes transposing the chromatin of the cells to a Tn5 transposon assembled with primer dimers and Tn5 transposase, cleaving the chromatin open sites of the cells, and ligating primer dimers to the chromatin open sites, wherein the primer dimers contain a handle sequence that subsequently encodes integration. c) Use reverse transcriptase and reverse transcription primers to perform reverse transcription on the transcriptome of cells to obtain transcriptome information of single cells; d) Using template-switching oligomers to carry out template-switching reactions; e) Subsequent encoding to obtain a transcriptome library and a chromatin open site library with cell coding, wherein the subsequent encoding includes single-cell isolation or coding integration of cells using different platforms or processes as needed, wherein the transcriptome library and the chromatin open site library from the same cell have the same cell coding or a combination of cell coding. f) Initial library expansion, which includes expanding the previously constructed library to increase the number of usable library fragments; g) Prepare chromatin open site libraries and transcriptome libraries for sequencing, including isolating the chromatin open site libraries and transcriptome libraries from the libraries amplified in the previous step. As shown in Figure 1, the template-converting oligomer contains a sequence ⑤ that is completely complementary to sequence ⑧ generated by the terminal transferase activity of reverse transcriptase, enabling the reverse transcriptase to continue sequence synthesis using the template-converting oligomer as a template. The template-converting oligomer also includes a partial structural region ④ located at the 5' end of sequence ⑤ and a handle sequence ③ located at the 5' end of the partial structural region ④ for subsequent coding integration. Furthermore, the template-converting oligomer includes a sequence ⑥ that is complementary to the partial structural region ④ to form a double-stranded structure with the template-converting oligomer. The partial structural region ④ in Figure 1 includes one or more functional regions, which are selected from unique molecular recognition signals, partial coding, and double-stranded complementary sequences. The method can link the coding to open sites on chromatin and / or the transcriptome while preserving the 5' information of the transcriptome.

2. The method of claim 1, wherein the single-cell suspension in step a) is not used or is fixed with a fixative before being used in subsequent steps.

3. The method of claim 2, wherein the fixative is formaldehyde.

4. The method of claim 2, wherein the fixation is terminated or not terminated and used in subsequent steps.

5. The method according to any one of claims 1 to 3, wherein the primer dimer, template conversion oligomer, and reverse transcription primer are modified.

6. The method of claim 5, wherein the primer dimer, template-conversion oligomer, and reverse transcription primer are phosphorylated and biotinylated.

7. The method according to any one of claims 1 to 4, wherein the sequence ⑥ of Figure 1 is 3' phosphorylated and the 3' phosphorylation modification can be removed in a subsequent reaction.

8. The method of claim 7, wherein, Elimination of 3' phosphorylation modification via polynucleotide kinase reaction.

9. The method according to any one of claims 1 to 4, wherein the template-converting oligomer is attached to the medium or is in a free state.

10. The method of claim 9, wherein the medium is a bead or a plate.

11. The method of claim 9, wherein the medium is a magnetized bead or a chip.

12. The method according to any one of claims 1 to 4, wherein the sequence ⑤ of Figure 1 is composed of DNA, RNA, locked nucleic acid, or a combination thereof.

13. The method of any one of claims 1 to 4, wherein the sequence ⑥ of Figure 1 is modified with 5' phosphorylation.

14. The method of any one of claims 1 to 4, wherein the initial library amplification is performed by a PCR reaction, and the primers used contain coding structures, are part of a cell coding combination, and / or contain codes for the sample source, experimental batch, or cell species.

15. The method according to any one of claims 1 to 4, further comprising amplifying and purifying the separated chromatin open site library and transcriptome library.

16. The method according to any one of claims 1 to 4, further comprising adding a sequencing universal adapter to the isolated chromatin open site library and / or adding a sequencing universal adapter to the isolated transcriptome library.

17. The method of any one of claims 1 to 4, wherein the transcriptome comprises mRNA encoding T cell receptor (TCR), B cell receptor (BCR) and / or guide RNA in a CRISPR system, and the transcriptome library comprises a TCR library, a BCR library and / or a guide RNA library.

18. A single-cell transcriptome and chromatin accessibility dual-omics sequencing library prepared by the method of any one of claims 1 to 17.

19. A method for sequencing a single-cell transcriptome and chromatin accessibility dual-omics sequencing library, comprising sequencing a chromatin open site library and a transcriptome library prepared by the method of any one of claims 1 to 17, respectively.

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