Method for preparing single cell multi-sample multi-body library and use thereof

Abstract

The present invention relates to a method for preparing a single cell multi-sample multi-body library and a use thereof. The present invention relates to a method whereby an epigenome and a transcriptome can be simultaneously analyzed at a single cell level, and the principle of regulating the epigenome and the transcriptome has been identified. Accordingly, the present invention makes it possible to reduce reagent costs by about 1 / 10 compared to the prior art and simultaneously analyze six or more samples, thus being economical and having the advantage of being able to contribute to high-tech genomic research. According to the present invention, six samples can be simultaneously analyzed, and ATAC as an epigenome and 5 prime RNA information as a transcript can be simultaneously collected and used to clearly identify the starting point of gene expression that immediately responds to epigenetic changes.

Description

Method for constructing a single-cell multisample multilayer library and its uses

[0001] The present invention relates to a method for producing a single-cell multi-sample multi-body library and its uses.

[0002] Single-cell analysis refers to a technique for analyzing genomics, transcriptomics, spatial transcriptomics, epigenomics, or proteomics at the single-cell level, unlike methods that analyze various cells simultaneously. A single cell refers to a single cell isolated from a cell population; since cellular heterogeneity exists even among cells derived from the same source, research at the single-cell level is crucial for accurate analysis. In particular, when dealing with stem cell research or diseases requiring the analysis of inherently heterogeneous cell populations, such as cancer, there is an increasing emphasis on the need to investigate molecular markers at the single-cell level rather than at the average level. Furthermore, advancements in single-cell analysis technology offer the potential to establish a systematic reference map for all human cells.

[0003] Projects utilizing single-cell analysis technology to analyze various diseased tissues are underway in multiple countries, and active efforts are being made in diagnosis, healthcare, and new drug development. While conventional bulk analysis methods have been highly effective in easily accumulating information related to specific omics domains, they have limitations in accurately identifying signals directly correlated with diseases because they only present the average values ​​of individual omics. However, analyzing single-cell multiples allows for the derivation of more complete information about each cell compared to focusing solely on individual omics. This enables the more precise identification of the complex interaction networks responsible for cellular functions. Furthermore, since cells derived from a single source generate diverse types and tissues are composed of such cell populations, accurate disease diagnosis or treatment is only possible by confirming and identifying the specificity of individual cells through single-cell multiple analysis. Additionally, single-cell multiple analysis possesses the characteristic of integrating and analyzing multi-layered omics information regarding specific cells to rapidly derive meaningful insights.

[0004] A multiplex library is an approach that integrates diverse omics information from biological data to understand and analyze the complex systems of living organisms. Omics is a collective term for various biological data, including genomics, transcriptomics, proteomics, and metabolomics. Omics technology is classified into genomics, which studies the relationships between genes through DNA sequencing; transcriptomics, which analyzes changes in gene expression through RNA expression profiling; proteomics, which studies the structure, function, and interactions of proteins; and metabolomics, which understands physiological states by analyzing changes in metabolites.

[0005] Because biological systems consist of complex interactions, it is often difficult to obtain sufficient insights from single omics data alone. Multiples are necessary from the perspective that understanding biological mechanisms requires simultaneously considering the impact of genetic variations on the transcriptome and proteome. There are various methods for integrating and analyzing multiples data. These include statistical modeling, machine learning techniques, and network analysis; through these methods, it is possible to identify correlations between multiple omics data and derive new biological insights. Furthermore, multiples analysis can be utilized in disease research by contributing to understanding the pathogenesis of specific diseases and developing personalized therapies, and it can also contribute to drug development by designing more effective drugs through in-depth analysis of their mechanisms of action. Such multiples libraries play a crucial role in understanding the complexity of organisms by integrating and analyzing diverse biological data, thereby driving innovative advancements in life science research and medical applications.

[0006] Meanwhile, single-cell multiplex library information is a technology that is gaining attention among researchers worldwide as it enables research on the cellular diversity of human tissues. However, the most widely used commercial product, the multiome kit from 10X Genomics, costs nearly 10 million won per sample, making it difficult to easily apply to research. Therefore, this invention was conceived to overcome the high costs and limitations of analysis associated with the production of existing single-cell multiplex libraries, reduce analysis costs to one-tenth of the existing cost, and contribute to the study of the relationship between epigenetic information (ATAC) and 5-prime RNA.

[0007] Accordingly, the inventors have completed the present invention by developing an analysis method using a single-cell multiplex library capable of simultaneously collecting and analyzing ATAC (Assay for Transposase-Accessible Chromatin using sequencing) and transcriptome (RNA) information as epigenomics within the same cell. Therefore, by enabling the clear identification of the starting point of gene expression within the same cell, this method can accelerate basic science research and be widely utilized for multiplex analysis.

[0008] The object of the present invention is to provide a method for generating a multiplex library capable of simultaneous analysis of multiplexes within a single cell, comprising: 1) a step of extracting a nucleus from a biological sample; 2) a step of treating the nucleus with a first transposase to insert a barcode into open DNA; 3) a step of treating with a reverse transcriptase to synthesize DNA complementary to mRNA, performing reverse transcription, and generating an RNA-DNA hybrid; 4) a step of generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and 5) a step of loading the nucleus, which has been pretreated by amplifying the DNA and RNA reads, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0009] Another objective of the present invention is to provide a single-cell multi-sample multi-sequencing library production kit utilizing the multi-sample library production method described above.

[0010] Another objective of the present invention is to provide a single-cell multi-sample multi-sequencing analysis kit utilizing the method for generating a multi-sample library.

[0011] Another objective of the present invention is to provide a single-cell mutation-based cancer targeted therapy candidate analysis kit utilizing the method for generating the multiplex library described above.

[0012] Another objective of the present invention is to provide a library generation system capable of simultaneous analysis of multiples within a single cell, comprising: an extraction unit for extracting a nucleus from a biological sample; an insertion unit for treating the nucleus with a first transposase to insert a barcode into open DNA; a hybrid generation unit for synthesizing DNA complementary to mRNA by treating with a reverse transcriptase to perform reverse transcription and generate an RNA-DNA hybrid; a library generation unit for generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and a read generation unit for loading the nucleus, which has been pre-treated with the DNA and RNA reads amplified, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0013] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0014] The present invention provides a method for generating a multiplet library capable of simultaneous analysis of multiplets within a single cell, comprising: 1) a step of extracting a nucleus from a biological sample; 2) a step of treating the nucleus with a first transposase to insert a barcode into open DNA; 3) a step of treating with a reverse transcriptase to synthesize DNA complementary to mRNA, performing reverse transcription, and generating an RNA-DNA hybrid; 4) a step of generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and 5) a step of loading the nucleus, which has been pretreated by amplifying the DNA and RNA reads, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0015] In addition, the present invention provides a single-cell multi-sample multi-sequencing library production kit utilizing the above-described multi-sample library production method.

[0016] In addition, the present invention provides a single-cell multi-sample multi-sequencing analysis kit utilizing the multi-sample library generation method described above.

[0017] In addition, the present invention provides a single-cell mutation-based cancer targeted therapy candidate analysis kit utilizing the above-described multiplex library generation method.

[0018] In addition, the present invention provides a library generation system capable of simultaneous analysis of multiples within a single cell, comprising: an extraction unit for extracting a nucleus from a biological sample; an insertion unit for treating the nucleus with a first transposase to insert a barcode into open DNA; a hybrid generation unit for synthesizing DNA complementary to mRNA by treating with a reverse transcriptase to perform reverse transcription and generate an RNA-DNA hybrid; a library generation unit for generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and a read generation unit for loading the nucleus, which has been pre-treated with the DNA and RNA reads amplified, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0019] The present invention relates to a method capable of simultaneously analyzing the epigenome and transcriptome at the single-cell level, thereby elucidating the principles of epigenome and transcriptome regulation. Accordingly, it offers economic advantages by reducing reagent costs to about one-tenth of existing methods and enabling the simultaneous analysis of six or more samples, and can contribute to advanced genomic research. According to the present invention, six samples can be analyzed simultaneously, and ATAC as the epigenome and 5-prime RNA as the transcriptome can be collected simultaneously to clearly identify the starting point of gene expression that responds immediately to epigenetic changes.

[0020] FIG. 1 is a schematic diagram illustrating the operating principle of a method for simultaneous analysis of epigenome and transcriptome according to an embodiment of the present invention. Each step and the characteristics of each step are as follows. (1) Step of binding of Tn5 transposase (S7) to open chromatin (epigenome): When the Tn5 (S7) enzyme is treated to a sample (nucleus), the Tn5 (S7) enzyme enters the nucleus and performs tagmentation on a loosely open site in the chromatin. Through this, the barcode possessed by the Tn5 (S7) enzyme is inserted into the open chromatin (DNA). (2) Step of reverse transcription (RT) using oligo dT and TSO (template switch oligo) primers: A step of synthesizing DNA complementary to RNA through RT (reverse transcription) enzyme treatment. By utilizing the characteristic of mRNA having polyA, reverse transcription is carried out using a polyT primer complementary thereto. When mRNA is synthesized to the end, the RT (reverse transcriptase) enzyme used in this invention attaches three cytosine bases (CCC) to the end, and a TSO primer containing GGG complementarily to it is attached. The TSO primer is assigned a sample-specific barcode. By assigning the sample barcode to the TSO primer, the design ultimately enables the production of a 5-prime RNA library. Furthermore, when targeting 5-prime RNA, unlike existing methods targeting 3-prime RNA, the first DNA strand produced is utilized; consequently, there are no broken sequences, and since the DNA is entirely single-stranded, no additional RT step is required, thereby simplifying the process. This increases nuclear yield and improves experimental convenience compared to conventional experiments.(3) Tn5 transposase (S5) binding step to open chromatin and DNA-RNA hybrid (epigenetics, transcriptome): This is a process in which Tn5 (S5) enzyme treatment is performed simultaneously on two types of molecules: chromatin (DNA) and RNA-DNA hybrid state (RNA) generated by reverse transcriptase action. The Tn5 (S5) enzyme enters the nucleus and performs tagmentation on the loosely open areas of the chromatin. Simultaneously, tagmentation is performed on the RNA-DNA hybrid molecule to lay the foundation for generating two libraries at the same time. (4) Droplet step: This is the state before loading the nucleus, pretreated using the 10x Genomics scATAC kit, into a droplet. In order for the final library to be generated, the necessary DNA and RNA reads must be amplified by SBS primers. (5) Step after primary expansion in GEM (using SBS primers): Nuclei pretreated using the 10x Genomics scATAC kit are loaded into droplets, and after the first PCR process using SBS primers, sequence reads are generated that can attach to beads containing nuclei within the droplets. Subsequently, the desired reads are generated by attaching to beads containing cell barcodes. Then, DNA and RNA libraries are generated from a sample through the library generation process.

[0021] FIG. 2 is a flowchart illustrating a method for simultaneous analysis of epigenetics and transcriptomes according to one embodiment of the present invention.

[0022] Figure 3 shows the analysis results by a library produced by a method for generating a multibody library according to an embodiment of the present invention.

[0023] Unless otherwise specifically defined in this invention, all scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the art to which this invention pertains. When a part of this invention is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0024]

[0025] The present invention will be described in more detail below.

[0026]

[0027] The present invention provides a method for generating a multiplet library capable of simultaneous analysis of multiplets within a single cell, comprising: 1) a step of extracting a nucleus from a biological sample; 2) a step of treating the nucleus with a first transposase to insert a barcode into open DNA; 3) a step of treating with a reverse transcriptase to synthesize DNA complementary to mRNA, performing reverse transcription, and generating an RNA-DNA hybrid; 4) a step of generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and 5) a step of loading the nucleus, which has been pretreated by amplifying the DNA and RNA reads, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0028] The inventors have made diligent research efforts to produce a multi-ome library capable of simultaneously verifying epigenetics (ATAC) and transcriptomes (5-prime RNA) in a single cell. As a result, they have perfected an economical library production technology that can perform more than six samples at once and reduce the reagent cost per sample by about one-tenth compared to existing methods.

[0029] The present invention significantly reduces analysis costs compared to existing commercial products (10x Genimics scATAC kit), by attaching molecular barcodes to six samples (nuclei) before using the commercial product, thereby producing a single-cell multiplex library of a total of six samples at once.

[0030] In the present invention, “biological sample” means any substance, biological body fluid, tissue, or cell obtained from or derived from an individual, e.g., whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchi It may include, but is not limited to, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, or cerebrospinal fluid.

[0031] In the present invention, "multiome" is a term including transcriptome, epigenome, genome, spatial transcriptome, and proteome, and may be one or more selected from the group consisting of transcriptome, epigenome, genome, spatial transcriptome, and proteome.

[0032] In the present invention, the term “transcriptome” refers to the sum of all expressed RNAs. Transcriptomics research is an omics field that comprehensively studies gene expression, encompassing not only coding genes involved in protein coding but also non-coding RNAs, such as microRNAs, that are not involved in coding. It received significant attention in the early 2000s with the introduction of microarray technology, but since 2010, with the introduction of NGS technology, it has been replaced by RNA sequencing. Recently, with the introduction of single-cell transcriptomics technology, it has become possible to analyze gene expression by cell type within a tissue, and it is being actively utilized in research on disease mechanisms. Transcriptomics research is the most widely utilized field in the life sciences; in particular, it can be applied to cell differentiation, stem cells, tumorigenesis, gene regulation, or biomarker discovery, and is utilized not only for expression profiling but also for alternative splicing analysis and RNA editing analysis.

[0033] In the present invention, the "epigenome" includes chemical changes in the genome and is a factor that regulates gene expression through structural changes such as DNA methylation and histone modification. The aforementioned epigenomics research is a field that studies epigenetic modifications of the genome and refers to a research field that interprets phenotypic characteristics resulting from acquired influences. It primarily studies the association between changes in DNA methylation and histone modification and genetic regulation, and based on this, it is possible to interpret phenotypic plasticity, which cannot be interpreted solely by genetic composition. Experimental techniques such as bisulfite sequencing to measure DNA methylation, chromatin immunoprecipitation (ChIP)-sequencing to identify histone modification marking regions, and DNase / ATAC-seq to analyze open / closed chromatin regions are utilized. Recently, epitranscriptomics research, which studies transcript modifications such as m6A, is also actively underway, and based on this, the causes of various biological phenomena such as cell differentiation, developmental processes, and tumor formation are being identified.

[0034] In this invention, the term “Genome” refers to the entire set of DNA containing all genetic information of a specific organism. The aforementioned genomics research refers to the field of omics that studies the genetic composition of each individual using next-generation sequencing (NGS) techniques. It includes whole-genome sequencing (WGS), which measures the entire genome; whole-exome sequencing (WES), which identifies only protein-coding regions; and single nucleotide polymorphism (SNP) genotyping research. Research on genomic medicine is active, utilizing genomic information generated by the above technologies to identify causal variants of diseases through genome-wide association studies (GWAS), predict genetic risk of diseases based on this information, and predict drug response. Furthermore, this information is being utilized in various life science fields such as population genetics, evolutionary biology, and synthetic biology.

[0035] In the present invention, the “spatial transcriptome” is a concept representing the spatial distribution of RNA molecules within a tissue or cell. Spatial transcriptome research is a technology capable of simultaneously observing positional information and gene expression patterns of each cell within the spatial context of a tissue section, and refers to a field that allows for the study of cell distribution and inter-cell interactions within the actual spatial context of a tissue. With the development of high-resolution technology capable of distinguishing from dozens of cells down to individual cell compartments, it is being utilized particularly in research aimed at understanding the microenvironment within cancer tissues.

[0036] In the present invention, the term “proteome” refers to the sum of all proteins expressed in a specific cell, tissue, or organism. Proteomics research is an omics research field that analyzes the expression or modification of proteins constituting living organisms, and it refers to a field capable of comprehensively studying proteins that directly mediate physiological functions in the human body. As a core pillar of functional genomics research, which has garnered significant attention since the Human Genome Project, it enables the analysis of post-translational modification regulation or protein isoforms that cannot be identified through genomic or transcriptomic analysis, and allows for the discovery of various biomarkers used in clinical settings. Due to the inherent complexity of proteins, proteomics requires various analytical techniques, and technologies such as antibody-based affinity proteomics and mass spectrometry-based shotgun proteomics are widely utilized.

[0037] In the present invention, “Library” refers to a collection of DNA molecules sufficient to contain all single genes present in a specific organism.

[0038] In the present invention, TSO (Template-switching-oligo) refers to an oligo that hybridizes to an untemplated C nucleotide added by a reverse transcriptase during reverse transcription.

[0039] In the present invention, a "primer" is a fragment that recognizes a target gene sequence and comprises a forward and a reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that it amplifies only the target sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0040] In the present invention, multi-omics analysis is a cutting-edge research technique that measures the total changes of various biomolecules within a cell and performs integrated analysis. It is particularly highly utilized in basic life science and clinical research, such as interpreting gene functions and identifying disease mechanisms. With the recent introduction of next-generation sequencing (NGS) techniques, it has become possible to generate large-scale multi-omics data at low cost, and as a result, multi-omics big data is becoming widely available.

[0041] In the present invention, the simultaneous analysis of the multiples may involve performing at least two analyses selected from the group consisting of transcriptome, epigenome, genome, spatial transcriptome, and proteome simultaneously.

[0042] In the present invention, the single cell may be a primary cell. The single cell may be isolated by flow cytometry.

[0043] In the present invention, the first transposase may be Tn5 transposase (S7). The first transposase may be an enzyme that measures the degree of condensation of chromatin corresponding to epigenetic information and may bind to relatively less condensed chromatin.

[0044] In the present invention, the second transposase may be Tn5 transposase (S5). The second transposase is an enzyme that simultaneously measures epigenetic and transcriptomic information, and binds to relatively less condensed chromatin and simultaneously binds to RNA-DNA hybrids.

[0045] In the present invention, the reverse transcriptase may induce the binding of a TSO primer to mRNA to impart a barcode to the open DNA. It may be designed to produce a 5-prime RNA library using the barcode. The barcode may include a cell barcode or a sample barcode.

[0046] In addition, the present invention provides a single-cell multi-sample multi-sequencing library production kit utilizing the above-described multi-sample library production method.

[0047] In addition, the present invention provides a single-cell multi-sample multi-sample sequencing library analysis kit utilizing the method for generating the multi-sample library described above.

[0048] In addition, the present invention provides a single-cell mutation-based cancer targeted therapy candidate analysis kit utilizing the above-described multiplex library generation method.

[0049] In addition, the present invention provides a library generation system capable of simultaneous analysis of multiples within a single cell, comprising: an extraction unit for extracting a nucleus from a biological sample; an insertion unit for treating the nucleus with a first transposase to insert a barcode into open DNA; a hybrid generation unit for synthesizing DNA complementary to mRNA by treating with a reverse transcriptase to perform reverse transcription and generate an RNA-DNA hybrid; a library generation unit for generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and a read generation unit for loading the nucleus, which has been pre-treated with the DNA and RNA reads amplified, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read.

[0050] Meanwhile, since the corresponding characteristics can be substituted in the aforementioned section, their description is omitted.

[0051]

[0052] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0053]

[0054] The experimental process and experimental results for simultaneously analyzing the epigenome and transcriptome according to one embodiment of the present invention were confirmed as follows.

[0055]

[0056] Example 1. Process for constructing a library capable of simultaneously analyzing the epigenome and transcriptome according to the present invention

[0057]

[0058] 1.1. Tn5 transposase (S7) binds to open chromatin

[0059]

[0060] Tn5 buffer (38.8 mM Tris-Acetate, 77.6 mM Potassium Acetate, 11.8 mM Magnesium Acetate, 18.8 mM DMF (Dimethylformamide), 0.5% 1X Protease Inhibitor Cocktail, RNase Inhibitor 1.6 U, 1X RSB buffer (10 mM Tris-HCl_pH7.5, 10 mM NaCl, 3 mM MgCl2)) was added to each of at least 6 samples (nuclei) up to 28.5 µl, and a Tn5 and Tn7 mix was added (total 30 µl). The mixture was reacted for 30 minutes at a speed of 500 rpm in a 30°C incubator. After the reaction was complete, the samples were transferred to ice, 2X Stop Buffer (20mM EDTA_pH 8.0, 2% Bovine Serum Albumin, 1X RSB Buffer) was added, and the mixture was mixed by pipetting 2-3 times. 170 µl of 1X RSB-T Buffer (1X RSB, 0.01% Triton X-100, 0.2 U / µl RNase Inhibitor, 1mM DTT (dithiothreitol)) was added to each sample and mixed 2-3 times with a pipette, followed by centrifugation at 500g, 4°C for 5 minutes. After removing the supernatant, the process was repeated two more times. During the final centrifugation, only about 10 µl of sample remained in the tube.

[0061]

[0062] 1.2. Reverse Transcription (RT) using Oligo dT and TSO (template switch oligo) Primers

[0063]

[0064] 20 µl of RT (Reverse Transcription) buffer (50 mM Tris-HCl_pH 8.0, 75 mM NaCl, 3 mM MgCl2, 10 mM DTT, 12% PEG8000, 0.5 mM dNTP, 1.2 U / µl RNase Inhibitor, 10 U / µl Maxima H minus RTase, 5 mM TSO oligo, 5 mM Poly T oligo) was added to each sample. After mixing 5–7 times with a pipette, the RT reaction was carried out (50°C 10 min, [8°C 12 sec, 15°C 45 sec, 20°C 45 sec, 30°C 30 sec, 42°C 2 min, 50°C 3 min] repeated 3 times, 50°C 5 min, 4°C). After adding 100 µl of 1X RSB-T buffer to each sample and mixing 2–3 times with a pipette, the samples were combined into a single tube. The supernatant was removed by centrifugation at 500g, 4°C for 7 minutes, and the process was repeated two more times (5 min centrifugation per repetition). During the final centrifugation, approximately 50 µl of sample remained in the tube, and the number of nuclei was counted using the yoyo-1 DNA staining reagent (mixed 1 µl of sample with 9 µl of yoyo-1 reagent and examined under a microscope).

[0065]

[0066] 1.3. Tn5 transposase (S5) binds to open chromatin and DNA-RNA hybrid

[0067]

[0068] 100 µl of Tn5(S7) buffer (38.8 mM Tris-Acetate, 77.6 mM Potassium Acetate, 11.8 mM Magnesium Acetate, 18.8 mM DMF (Dimethylformamide), 0.5% 1X Protease Inhibitor Cocktail, 0.4 µl / 100 µl Tn5(S7)) was added per 100,000 nuclei. The reaction was carried out in a 37°C incubator at 500 rpm for 30 minutes. After the reaction was complete, the sample was transferred to ice, 2X Stop buffer (20 mM EDTA_pH 8.0, 2% Bovine Serum Albumin, 1X RSB buffer) was added, and the mixture was mixed by pipetting 2-3 times. After removing the supernatant under conditions of centrifugation at 500 g, 4°C for 5 minutes, the process was repeated two more times. During the final centrifugation, only about 16 µl of the sample remained in the tube. The number of nuclei was confirmed using the yoyo-1 DNA staining reagent (mixed 1 µl of sample with 9 µl of yoyo-1 reagent and examined under a microscope).

[0069]

[0070] 1.4. Droplet Step

[0071]

[0072] A mix was prepared in advance by adding primers specific to the present invention to the 10x Genomics kit (Barcoding Reagent B 56.5 µl, Reducing Agent B 1.5 µl, Barcoding Enzyme 2 µl, 100 µM Blocking Oligo 0.75 µl, 100 µM SBS primer for RNA 0.75 µl). The remaining steps were performed identically to the procedures provided by the 10x Genomics kit manufacturer (however, the number of linear amplification cycles was changed from the original 12 cycles to 4–10 cycles).

[0073]

[0074] 1.5. After primary expansion within GEM (using SBS primer)

[0075]

[0076] After extracting DNA from the droplet, PCR mix (NEBNext HF 2X PCR Master Mix 50 µl, 10 µM P5 primer 5 µl, 10 µM SBS for ATAC primer 5 µl, 10 µM SBS for RNA primer 5 µl) was added and PCR was performed (98°C 1 min, [98°C 20 sec, 63°C 20 sec, 72°C 20 sec] repeated 5 times, 72°C 1 min, 4°C). Afterward, DNA was extracted, and the sample was divided in half to construct RNA and ATAC libraries, respectively, through PCR. ATAC PCR was performed using a mix of (NEBNext HF 2X PCR Master Mix 50 µl, 10 µM P5 primer 5 µl, 10 µM SBS for ATAC primer 5 µl) (98°C 1 min, [98°C 20 sec, 63°C 20 sec, 72°C 20 sec] repeated 5 times, 72°C 1 min, 4°C). RNA PCR was performed using a mix of (NEBNext HF 2X PCR Master Mix 50 µl, 10 µM P5 primer 5 µl, 10 µM SBS for RNA primer 5 µl) (98°C 1 min, [98°C 20 sec, 63°C 20 sec, 72°C 20 sec] repeated 5 times, 72°C 1 min, 4°C).

[0077]

[0078] Example 2. Results of simultaneous analysis of epigenome and transcriptome according to the present invention

[0079]

[0080] The analysis results by the library produced by the multibody library generation method according to the present invention are shown in Figure 3.

[0081]

[0082] As a result of measuring the DNA quantity and length distribution of each library using a DNA analyzer, it was confirmed that the libraries possess a length distribution and shape suitable for NGS sequencing libraries.

[0083]

[0084] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0085]

[0086] The national research and development projects that supported this invention are as follows.

[0087]

[0088] [Project No.] NRF-2022M3A9D3016848

[0089] [Ministry Name] Ministry of Science and ICT

[0090] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0091] [Research Project Name] Biomedical Technology Development Project - Development of Immune Mechanism Control Technology

[0092] [Project Title] Establishment of a Metaplatform for Discovering Molecular Targets for Disease Treatment through the Construction of a Multilayered Single-Cell Map of Immune Diseases

[0093] [Contribution Rate] 1 / 2

[0094] [Name of Project Performing Organization] Seoul National University Industry-Academic Cooperation Foundation

[0095] [Research Period] 2022.04.01-2024.12.31

[0096]

[0097] [Project ID] 1345374154

[0098] [Assignment No.] 2021R1A6C101A445

[0099] [Ministry Name] Ministry of Education

[0100] [Project Management (Specialized) Agency Name] Korea Basic Science Institute

[0101] [Research Project Name] Basic Science Research Capacity Enhancement Project

[0102] [Project Title] Support Project for Establishing a Core Research Support Center (Genomic Medicine Research Support Center)

[0103] [Contribution Rate] 1 / 2

[0104] [Name of Project Performing Organization] Seoul National University Industry-Academic Cooperation Foundation

[0105] [Research Period] 2021.06.01-2027.02.28

Claims

1. A method for generating a multiplex library capable of simultaneous analysis of multiplexes within a single cell, wherein 1) A step of extracting the nucleus from a biological sample; 2) A step of treating the nucleus with the first transposase to insert a barcode into the open DNA; 3) A step of synthesizing DNA complementary to mRNA by treating with reverse transcriptase to proceed with reverse transcription and generating an RNA-DNA hybrid; 4) a step of generating a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; and 5) a step of loading a pre-treated nucleus, in which the above DNA and RNA reads have been amplified, into a droplet, generating a sequence read capable of attaching to a bead within the droplet, and attaching to a bead containing a cell barcode to generate a desired read; comprising a method for generating a library.

2. In Paragraph 1, A method characterized in that the above multiple is one or more selected from the group consisting of transcriptome, epigenome, genome, spatial transcriptome, and proteome.

3. In Paragraph 1, A method characterized by the simultaneous analysis of the above multiples being performed simultaneously by at least two analyses selected from the group consisting of transcriptome, epigenome, genome, spatial transcriptome, and proteome.

4. In Paragraph 1, A method characterized in that the above single cell is a primary cell.

5. In Paragraph 1, A method characterized by isolating the above single cell by flow cytometry.

6. In Paragraph 1, A method characterized in that the first transposase is a Tn5 transposase (S7).

7. In Paragraph 1, A method characterized by the first transposase agent measuring the degree of condensation of chromatin corresponding to epigenetic information.

8. In Paragraph 1, A method characterized in that the second transposase is a Tn5 transposase (S5).

9. In Paragraph 1, A method characterized by the above-mentioned second transposase simultaneously measuring epigenetic and transcriptomic information.

10. In Paragraph 1, A method characterized by the above reverse transcriptase inducing the binding of a TSO primer to mRNA to impart a barcode to open DNA.

11. In Paragraph 1, A method characterized by designing a 5-prime RNA library to be produced by the above-mentioned barcode.

12. In Paragraph 1, A method characterized in that the above barcode includes a cell barcode or a sample barcode.

13. A single-cell multi-sample multiplex sequencing library production kit utilizing the method for producing a multiplex library according to any one of claims 1 to 12.

14. A single-cell multi-sample multiplex sequencing analysis kit using the method for generating a multiplex library of any one of claims 1 to 12.

15. A single-cell mutation-based cancer targeted therapy candidate analysis kit utilizing the method for generating a multiplex library according to any one of claims 1 to 12.

16. A multibody library generation system capable of simultaneous analysis of multiples within a single cell, An extraction unit for extracting nuclei from biological samples; An insertion part that inserts a barcode into open DNA by treating the nucleus with a first transposase; A hybrid production unit that synthesizes DNA complementary to mRNA by treating with reverse transcriptase to proceed with reverse transcription and generates an RNA-DNA hybrid; A library production unit that generates a simultaneous library of the open DNA and RNA-DNA hybrid using a second transposase; A library generation system comprising: a read generation unit that loads a pre-processed nucleus, in which the above DNA and RNA reads are amplified, into a droplet, generates a sequence read capable of attaching to a bead within the droplet, and attaches to a bead containing a cell barcode to generate a desired read.

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