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WO2021189679A1 - Méthode de construction d'une banque de séquençage de transcriptome de cellule unique et son utilisation - Google Patents

Méthode de construction d'une banque de séquençage de transcriptome de cellule unique et son utilisation Download PDF

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Publication number
WO2021189679A1
WO2021189679A1 PCT/CN2020/096320 CN2020096320W WO2021189679A1 WO 2021189679 A1 WO2021189679 A1 WO 2021189679A1 CN 2020096320 W CN2020096320 W CN 2020096320W WO 2021189679 A1 WO2021189679 A1 WO 2021189679A1
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Prior art keywords
sequencing
cell
transposase
library
reverse transcription
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Chinese (zh)
Inventor
万瑛
于海礼
仇鑫
陈钢
郑子寒
倪青山
周逸文
许昊
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Army Medical University
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Army Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the field of biotechnology, in particular to the field of single-cell sequencing, and in particular to a method for constructing a single-cell transcriptome sequencing library and its application.
  • RNA-sequencing (RNA-seq) technology has developed rapidly and has become an indispensable tool for differential gene expression and mRNA splicing analysis, and has been widely used in the field of biomedicine.
  • Most of the existing RNA-seq methods are based on cell populations, and the results only reflect the average expression level of genes in a multi-cell population, and cannot show differences in gene expression in each cell.
  • the recently developed single-cell RNA-seq is a new technology for sequencing RNA at the level of individual cells. It can not only effectively solve the heterogeneity of tissue samples and the transcriptome heterogeneity of cell populations masked by conventional RNA-seq. Sexual problems also provide a new research direction for analyzing the behavior, mechanism and relationship of single cells, and become a new way of single cell research nowadays.
  • the method of single-cell RNA library construction usually requires pre-amplification of a very small amount of RNA in a single cell, such as reverse transcription of RNA into cDNA, and then adding known sequences to both ends for PCR amplification, or using IVT technology performs in vitro transcription and amplification of cDNA, but neither of these two methods of library construction can avoid the bias introduced by PCR amplification, causing problems such as uneven transcriptome coverage, large background noise, and inaccurate quantification; similarly, these methods will Gene dropout is caused by the loss of some transcripts during the amplification and capture process. Especially for genes with low and medium expression levels, the dropout phenomenon will be more obvious. These genes with low and medium expression levels are useful for analyzing the co-expression of genes and genes.
  • the present invention provides a method for constructing a single-cell transcriptome sequencing library and its application, which can quickly and effectively construct a single-cell transcriptome library.
  • the present invention uses Tn5 transposase to directly perform DNA on double-stranded cDNA. Fragmentation and ligation reactions do not require PCR pre-amplification of cDNA, and can quickly and efficiently construct single-cell transcriptome sequencing libraries. This method can detect more genes, effectively reduce the proportion of low and medium abundance genes dropout, obtain more accurate transcriptome information, and reduce the time and cost of library construction.
  • the first aspect of the present invention provides a method for constructing a single-cell transcriptome sequencing library, which includes the following steps:
  • the single cell is an in vitro cultured cell, tissue cell, and free cell in body fluid; the single cell is derived from, but not limited to, one of human, rat, and mouse tissues; The single cells are sorted into the lysis solution by flow cytometry and then lysed on ice for 15 minutes to fully lyse the cells.
  • the reverse transcription primer is a fixed structure of TTTTTTTTTTTTTTTTTTTTTTTTTTTTVN from 5'to 3', and the fixed structure is used to specifically bind to the poly(A) tail of the mRNA to ensure reverse transcription Specificity.
  • the specific sequence and length range of the above-mentioned reverse transcription primers those skilled in the art can design and use them according to actual needs.
  • the reverse transcriptase used in reverse transcription is selected from one of Superscript II reverse transcriptase, Superscript III reverse transcriptase, or Superscript IV reverse transcriptase.
  • step (c) the second-strand cDNA synthesis reagent used is NEBNext Ultra II Non-Directional RNA Second Strand Module kit from New England Biolabs.
  • the Tn5 transposase is In-House Tn5 transposase
  • the preparation method of the In-House Tn5 transposase is: Tn5 recombinant plasmid transforms bacteria, induces expression by IPTG, and disrupts bacteria
  • the Tn5 transposase protein was purified using a Ni column; after assembly, it was transferred to a protein ultrafiltration concentration tube for ultrafiltration purification to obtain Tn5 transposase.
  • the In-House Tn5 transposase obtained by the above method has the characteristics of high purity and high activity, and high efficiency of library construction.
  • the primer sequences used in PCR enrichment of the template are the Read1 and Read2 sequences common to the Illumina sequencing platform.
  • the Read1 sequence is shown in SEQ ID NO: 21, and the Read 2 sequence is shown in SEQ ID NO: 22. Show.
  • the enriched template is purified by magnetic beads and then used for subsequent Index PCR.
  • the magnetic beads used can be Vazyme’s VAHTS DNA purification magnetic beads or the same type of magnetic beads.
  • the beads and the enriched template are purified at a ratio of 1:1.
  • one P5 and one P7 form a pair of primers, and the primers are adapted to the Illumina sequencing platform for sequencing.
  • the primer sequences of P5 and P7 are as follows:
  • Index is a nucleic acid sequence of 8 bp in length.
  • specific sequence of Index those skilled in the art can design more types for use according to actual needs.
  • the second aspect of the present invention provides the application of the above-mentioned method for constructing a single-cell transcriptome sequencing library in single-cell sequencing.
  • the third aspect of the present invention provides a single-cell sequencing method, which adopts the above-mentioned single-cell transcriptome sequencing library construction method to construct a single-cell sequencing library, and then uses a high-throughput sequencing platform for sequencing.
  • the high-throughput sequencing platform is an Illumina sequencing platform.
  • the fourth aspect of the present invention provides the method for constructing the single-cell transcriptome sequencing library or the application of the single-cell sequencing method in the preparation of detection kits, detection devices or detection systems for developmental research or cancer research.
  • the method for building a single-cell transcriptome database and its application of the present invention have the following beneficial effects:
  • the scSTAT-seq Single-cell Streamlined Transcription And Tagmentation Sequencing
  • the scSTAT-seq Single-cell Streamlined Transcription And Tagmentation Sequencing
  • Tn5 transposase to directly cut double-stranded cDNA without pre-PCR on the cDNA template.
  • Amplification can effectively reduce library preference and obtain more accurate transcriptome information; use In-House Tn5 transposase to synchronize DNA interruption and adaptor connection, which can reduce the amount of starting template and sample processing time, simplifying complex
  • the experimental operation can effectively improve the efficiency of library construction and reduce the cost of library construction; this method has simple steps for library construction, can detect more genes, effectively reduces the proportion of low-to-medium abundance genes dropout, and more truly reflects the single-cell gene expression situation. It is conducive to more detailed research at the single-cell level, and is more in line with the requirements of the development of single-cell sequencing.
  • Fig. 1 shows a flowchart of a database construction according to an embodiment of the present invention.
  • Figure 2 shows a distribution diagram of library fragments according to an embodiment of the present invention.
  • Figure 3 shows a violin diagram of the gene number distribution between the embodiment of the present invention and the SMART-seq2 method.
  • Fig. 4 shows a sequencing saturation curve diagram comparing the embodiment of the present invention and the SMART-seq2 method.
  • Figure 5 shows a gene dropout diagram comparing the embodiment of the present invention and the SMART-seq2 method.
  • the library construction in the present invention is based on mouse mononuclear macrophage leukemia cells RAW 264.7.
  • a 96-well plate single-cell library construction is taken as an example. If more single-cell libraries need to be constructed, only more index PCR primers need to be designed and synthesized.
  • the method for constructing a single-cell transcriptome database of the present invention is described in detail through the following steps:
  • composition of the solution is as follows:
  • the reverse transcription primer is a fixed structure of TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN from 5'to 3', and the fixed structure is used to specifically bind to the poly(A) tail of mRNA to ensure the specificity of reverse transcription.
  • Tn5 transposase is the In-House Tn5 transposase prepared by the laboratory.
  • the preparation method of In-House Tn5 transposase is as follows: Tn5 recombinant plasmid is transformed into bacteria, induced by IPTG and expressed, the bacteria are disrupted, and Ni column is used Purify the Tn5 transposase protein; after assembling, transfer it to a protein ultrafiltration concentration tube for ultrafiltration purification to obtain high-activity and high-purity Tn5 transposase.
  • Fragment purification add 50 ⁇ l VAHTS DNA purification magnetic beads to each well, pipette and mix them evenly, and leave them at room temperature for 5 minutes. Place the sample on the magnetic stand for minutes and remove the supernatant. Wash twice with 100 ⁇ l 80% ethanol. Add 23 ⁇ l of nuclease-free water, use a pipette to pipette to mix, and place at room temperature for 5 minutes.
  • one P5 and one P7 form a pair of primers, which are adapted to the Illumina sequencing platform for sequencing, and 96 pairs of primers are used to index 96-well cells.
  • the primer sequences of P5 and P7 are as follows:
  • Index is a nucleic acid sequence with a length of 8 bp.
  • sequence of Index those skilled in the art can design more types for use according to actual needs.
  • the P5 and P7 primers come from the Illumina library building kit, and the P5 primers include P501, P502, P503, P504, P505, P506, P507, P508, and the sequences of P501 ⁇ P508 are as SEQ ID NO.1 ⁇ SEQ ID No. 8; P7 primer includes P701, P702, P703, P704, P705, P706, P707, P708, P709, P710, P711, P712, the sequence of P701 ⁇ P712 is as SEQ ID NO.9 ⁇ SEQ ID NO.20 Shown.
  • the library obtained above was sequenced using the Illumina X10 platform and compared with the SMART-seq2 library.
  • the sequencing results through bioinformatics analysis are shown in Figures 3, 4, and 5.
  • the primer sequences used above are shown in Table 2.
  • the scSTAT-seq Single-cell Streamlined Transcription And Tagmentation Sequencing
  • uses Tn5 transposase to directly digest double-stranded cDNA without Pre-amplification of cDNA template by PCR can effectively reduce library preference and obtain more accurate transcriptome information
  • use In-House Tn5 transposase to synchronize DNA interruption and adaptor connection which can reduce the amount of starting template and sample processing Time, simplify complex experimental operations, effectively improve the efficiency of library construction, and reduce the cost of library construction
  • this method has simple steps for library construction, can detect more genes, effectively reduces the proportion of low-to-medium abundance genes dropout, and more truly reflects single cells
  • Gene expression conditions are conducive to more detailed research at the single-cell level and more in line with the requirements of the development of single-cell sequencing.

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Abstract

L'invention concerne une méthode de construction d'une bibliothèque de séquençage de transcriptome de cellule unique et son utilisation. La méthode comprend les étapes suivantes consistant à : lyser une cellule unique triée dans une plaque de puits, et réaliser une synthèse de transcription inverse à l'aide d'une amorce de transcription inverse de façon à synthétiser un premier brin d'ADNc ; utiliser une réaction de synthèse de remplacement pour synthétiser un second brin d'ADNc ; fragmenter un ADNc double brin en utilisant une transposase Tn5 ; enrichir un matrice fragmentée par PCR ; et marquer le fragment enrichi à l'aide d'une PCR indexée, et réaliser une purification de manière à obtenir la bibliothèque de séquençage de transcriptome de cellule unique.
PCT/CN2020/096320 2020-03-27 2020-06-16 Méthode de construction d'une banque de séquençage de transcriptome de cellule unique et son utilisation Ceased WO2021189679A1 (fr)

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Cited By (7)

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CN115109835A (zh) * 2021-12-24 2022-09-27 广州表观生物科技有限公司 一种细胞外rna文库的构建方法及其应用
CN115386622A (zh) * 2022-10-26 2022-11-25 北京寻因生物科技有限公司 一种转录组文库的建库方法及其应用
CN115386624A (zh) * 2022-10-26 2022-11-25 北京寻因生物科技有限公司 一种单细胞全序列标记的方法及其应用
CN116103368A (zh) * 2022-07-29 2023-05-12 南方科技大学 一种3’mRNA测序文库的构建方法
CN116536394A (zh) * 2023-04-26 2023-08-04 青岛百创智能制造技术有限公司 一种海洋生物单细胞转录组文库的构建方法
WO2023155135A1 (fr) * 2022-02-18 2023-08-24 深圳华大生命科学研究院 Procédé de construction et procédé de séquençage pour banque de séquençage de transcriptome de cellule unique et kit de test pour préparer une banque de transcriptomes de cellule unique
WO2024250155A1 (fr) * 2023-06-05 2024-12-12 清华大学 Procédé de construction d'une banque de séquençage de cellules individuelles

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CN114107459B (zh) * 2021-11-11 2022-12-06 浙江大学 一种基于寡核苷酸链杂交标记的高通量单细胞测序方法
CN114350748A (zh) * 2021-12-30 2022-04-15 厦门大学 一种快速单细胞建库方法
CN116694730B (zh) * 2022-02-28 2025-02-14 南方科技大学 一种单细胞开放染色质和转录组共测序文库的构建方法
CN114596915B (zh) * 2022-03-03 2025-07-25 中国人民解放军陆军军医大学 基于模板序列和参考细胞的TCR β 高通量测序数据校正和标准化的方法
CN114854827A (zh) * 2022-05-18 2022-08-05 苏州极客基因科技有限公司 一种用于富集mRNA的磁珠复合物及其应用
CN114934096A (zh) * 2022-05-31 2022-08-23 上海市生物医药技术研究院 用于实施免疫组库测序的组合物及试剂盒和测序方法
CN116042772B (zh) * 2023-02-10 2025-07-29 上海立闻生物科技有限公司 针对少量来自于新鲜样本的肿瘤细胞全转录组测序的方法和试剂盒
WO2025129521A1 (fr) * 2023-12-20 2025-06-26 深圳华大生命科学研究院 Procédé de construction de bibliothèque de transcriptome basé sur amplification à déplacement multiple
CN117683866B (zh) * 2024-01-22 2024-08-06 湛江中心人民医院 检测细胞中dna的方法

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115109835A (zh) * 2021-12-24 2022-09-27 广州表观生物科技有限公司 一种细胞外rna文库的构建方法及其应用
WO2023155135A1 (fr) * 2022-02-18 2023-08-24 深圳华大生命科学研究院 Procédé de construction et procédé de séquençage pour banque de séquençage de transcriptome de cellule unique et kit de test pour préparer une banque de transcriptomes de cellule unique
CN116103368A (zh) * 2022-07-29 2023-05-12 南方科技大学 一种3’mRNA测序文库的构建方法
CN115386622A (zh) * 2022-10-26 2022-11-25 北京寻因生物科技有限公司 一种转录组文库的建库方法及其应用
CN115386624A (zh) * 2022-10-26 2022-11-25 北京寻因生物科技有限公司 一种单细胞全序列标记的方法及其应用
CN115386622B (zh) * 2022-10-26 2023-10-27 北京寻因生物科技有限公司 一种转录组文库的建库方法及其应用
CN116536394A (zh) * 2023-04-26 2023-08-04 青岛百创智能制造技术有限公司 一种海洋生物单细胞转录组文库的构建方法
WO2024250155A1 (fr) * 2023-06-05 2024-12-12 清华大学 Procédé de construction d'une banque de séquençage de cellules individuelles

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