WO2021189679A1 - Method for constructing single cell transcriptome sequencing library and use thereof - Google Patents

Abstract

Disclosed are a method for constructing a single cell transcriptome sequencing library and use thereof. The method comprises the following steps: lysing a sorted single cell in a well plate, and performing reverse transcription synthesis by using a reverse transcription primer so as to synthesize a first strand cDNA; using a replacement synthesis reaction to synthesize a second strand cDNA; fragmenting a double-stranded cDNA by using a Tn5 transposase; enriching a fragmented template by using PCR; and labeling the enriched fragment by using Index PCR, and performing purification so as to obtain the single cell transcriptome sequencing library.

Description

Method for constructing single-cell transcriptome sequencing library and its application Technical field

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.

Background technique

In the past ten years, 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.

At present, 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. Expression and the contribution of low- and medium-expressed genes to biological pathways are very important. Although most single-cell transcriptome sequencing methods such as SCRB-Seq and Drop-Seq can detect a large number of cells, they can only sequence the 3′ end of the transcript, and each cell can only detect a small number of genes ; Smart-Seq method can use template conversion to amplify full-length cDNA, increasing the number of gene detection, but the number of detection cells is small, the steps are complicated, and the cost is higher than other methods. In addition, the traditional methods of sonication and restriction fragmentation library construction require complicated steps such as end repair and addition of linkers to achieve library construction. In order to effectively obtain the accurate transcriptome characteristics of a single cell, it is necessary to develop a new single-cell transcriptome sequencing technology.

Summary of the invention

In view of the above problems, 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.

To achieve the above and other related purposes, the first aspect of the present invention provides a method for constructing a single-cell transcriptome sequencing library, which includes the following steps:

(a) Lyse the sorted single cells in the well plate;

(b) Reverse transcription using reverse transcription primers to synthesize the first strand cDNA;

(c) Synthesize the second strand cDNA using displacement synthesis reaction;

(d) Use Tn5 transposase to fragment the double-stranded cDNA template;

(e) Using PCR to enrich the fragmented template;

(f) Use Index PCR to label the enriched fragments, and obtain a single-cell transcriptome sequencing library that can be directly used for sequencing after the product is purified.

Further, in step (a), 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.

Further, in step (b), 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 the mRNA to ensure reverse transcription Specificity. Regarding 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.

Further, in step (b), 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.

Further, in step (c), the second-strand cDNA synthesis reagent used is NEBNext Ultra II Non-Directional RNA Second Strand Module kit from New England Biolabs.

Further, in step (d), the Tn5 transposase is In-House Tn5 transposase, and 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.

Further, in step (e), 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.

Furthermore, in step (e), 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.

Further, in step (f), 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:

P5:AATGATACGGCGACCACCGAGATCTACAC[Index]TCGTCGGCAGCGTC,

P7: CAAGCAGAAGACGGCATACGAGAT[Index]GTCTCGTGGGCTCGG,

Where Index is a nucleic acid sequence of 8 bp in length. Regarding the 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. Wherein, 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.

As mentioned above, the method for building a single-cell transcriptome database and its application of the present invention have the following beneficial effects:

Compared with the existing single-cell sequencing technology, the scSTAT-seq (Single-cell Streamlined Transcription And Tagmentation Sequencing) technology of the present invention utilizes 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.

Description of the drawings

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.

Detailed ways

The following describes the implementation of the present invention through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

It should be noted that the library construction in the present invention is based on mouse mononuclear macrophage leukemia cells RAW 264.7.

The main reagents, instruments and materials used in the present invention are shown in Table 1 below.

Table 1

The specific implementation process is as follows:

In this embodiment, 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:

1. Lyse cells

1. Prepare the solution according to the following composition and distribute the solution to 96 wells. The composition of the solution is as follows:

1％CA630----------------------------------------------- ---0.6μl

Rnase OUT ribonuclease inhibitor------------------------0.025μl

Nuclease-free water---------------------------------------------- --1.175μl

Deoxyribonucleoside triphosphate master mix---------------------------0.1μl

Reverse transcription primer (2μM)---------------------------------------0.1μl

Total product ----------------------------------------------- -------2μl

Among them, 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.

2. Resuspend RAW 264.7 cells in PBS solution, and sort single cells into 96-well plates by flow cytometry.

3. Lysis on ice for 15 minutes.

2. Reverse transcription

1. Prepare the solution and distribute the solution to 96 wells containing lysed cells. The composition of the solution is as follows:

Dithiothreitol (0.1M)----------------------------------------- -----0.2μl

Superscript III first chain buffer (5x)----------------------------0.8μl

Rnase OUT ribonuclease inhibitor (40units/ul)----------------0.2μl

SuperScript III reverse transcriptase (200units/ul)-----------------------0.2μl

Nuclease-free water---------------------------------------------- --------0.6μl

total capacity------------------------------------------------ ------------2μl

2. Perform reverse transcription reaction: 10 minutes at 25°C, 10 minutes at 35°C, 10 minutes at 37°C, 60 minutes at 50°C, 15 minutes at 70°C, and hold at 4°C.

3. Second-strand cDNA synthesis

1. Prepare a solution and distribute the solution to 96 wells containing reverse transcription products. The composition of the solution is as follows:

NEBNext Second Strand Synthesis Reaction Buffer---------------------------0.8μl

NEBNext Second Strand Synthetase Mixture--------------------------------0.4μl

Nuclease-free water---------------------------------------------- ----------2.8μl

total capacity------------------------------------------------ --------------4μl

2. Perform the second-strand cDNA synthesis procedure: 16°C for 60 minutes, and 4°C hold.

4. Fragmentation of double-stranded cDNA template

1. Prepare a solution and distribute the solution to 96 wells containing double-stranded cDNA. The composition of the solution is as follows:

5x reaction buffer--------------------------------------------4μl

Tn5 transposase---------------------------------------------- ---1μl

Nuclease-free water---------------------------------------------- ---7μl

total capacity------------------------------------------------ -------12μl

Among them, 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.

2. Perform fragmentation program: 55°C for 10 minutes, 10°C hold.

3. After completing the fragmentation procedure, immediately add 5μl 0.2% SDS to stop the reaction, pipette to mix evenly, and place it at room temperature for 5 minutes.

Five, PCR enrichment and fragmentation template

1. Prepare a solution and distribute the solution to 96 wells containing fragmented templates. The composition of the solution is as follows:

5xTruePrep buffer-----------------------------------------10μl

TruePrep Amplification Enzyme--------------------------------------------1μl

Read1 primer (1μM) (SEQ ID NO: 21)----------------------1μl

Read2 primer (1μM) (SEQ ID NO: 22)----------------------1μl

Nuclease-free water---------------------------------------------- ---12μl

total capacity------------------------------------------------ ---------25μl

2. Perform fragment PCR enrichment program: 72°C for 3 minutes, 98°C for 30 seconds, 25 cycles (98°C for 15 seconds, 60°C for 30 seconds, 72°C for 3 minutes), 72°C for 5 minutes, 4°C hold.

3. Fragment purification: add 50μl 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 100μl 80% ethanol. Add 23μl of nuclease-free water, use a pipette to pipette to mix, and place at room temperature for 5 minutes.

Six, Index PCR

1. Prepare a solution and distribute the solution to 96 wells containing magnetic beads purification templates. The composition of the solution is as follows:

KAPA Hot Start HiFi High Fidelity Enzyme Mix------------------------25μl

P5 primer (1μM) (SEQ ID NO: 1～8)-------------------------------1μl

P7 primer (1μM) (SEQ ID NO: 9-20)------------------------------1μl

total capacity------------------------------------------------ ----------------27μl

Among them, 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:

P5:AATGATACGGCGACCACCGAGATCTACAC[Index]TCGTCGGCAGCGTC,

P7: CAAGCAGAAGACGGCATACGAGAT[Index]GTCTCGTGGGCTCGG.

Where Index is a nucleic acid sequence with a length of 8 bp. Regarding the specific sequence of Index, those skilled in the art can design more types for use according to actual needs.

In this example, 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.

2. Perform Index PCR program: 95°C for 30 seconds, 15 cycles (98°C for 20 seconds, 60°C for 15 seconds, 72°C for 1 minute), 72°C for 5 minutes, 4°C hold.

3. Aspirate the Index PCR product in the 96-well plate and use the QIAquick PCR purification kit to purify it to obtain a sequencing library compatible with the Illumina sequencing platform.

7. Sequencing the constructed library.

1. After the library is purified, use Agilent Fragment Analyzer to detect the fragment distribution of the library, and the detection results are shown in Figure 2.

2. 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.

Table 2 Primer sequence list

As can be seen from Figure 3, an average of up to 9,000 genes can be found in RAW 264.7 cells, which is an average of 3,000 more than SMART-seq2.

It can be seen from Figure 4 that although the SMART-seq2 library (blue curve) reaches saturation at about 500,000 reads/cell, the scSTAT-seq library (red curve) reaches saturation later; but at the same depth, scSTAT-seq On average, the library detected about 2000 more genes than the SMART-seq2 library.

It can be seen from Figure 5 that the scSTAT-seq library (blue curve) showed a lower dropout level than the SMART-seq2 library (red curve) in the linear regression analysis.

In summary, compared with the existing single-cell sequencing technology, the scSTAT-seq (Single-cell Streamlined Transcription And Tagmentation Sequencing) technology of the present invention 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.

The above-mentioned embodiments only exemplarily illustrate the principles and effects of the present invention, but are not used to limit the present invention. Anyone familiar with this technology can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

A method for constructing a single-cell transcriptome sequencing library, which is characterized in that it comprises the following steps: (a) Lyse the sorted single cells in the well plate; (b) Reverse transcription using reverse transcription primers to synthesize the first strand cDNA; (c) Synthesize the second strand cDNA using displacement synthesis reaction; (d) Use Tn5 transposase to fragment the double-stranded cDNA template; (e) Using PCR to enrich the fragmented template; (f) Use Index PCR to label the enriched fragments, and obtain a single-cell transcriptome sequencing library that can be directly used for sequencing after the product is purified. The method according to claim 1, characterized in that: in step (b), the reverse transcription primer is a fixed structure of TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN in order from 5'to 3', and the fixed structure is used to interact with the poly(A ) Tail binds specifically to ensure the specificity of reverse transcription. The method according to claim 1, characterized in that: in step (b), the reverse transcriptase used in reverse transcription is selected from one of Superscript II reverse transcriptase, Superscript III reverse transcriptase, and Superscript IV reverse transcriptase. kind; And/or, in 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 method according to claim 1, wherein in step (d), the Tn5 transposase is In-House Tn5 transposase, and the preparation method of the In-House Tn5 transposase is: Tn5 recombination The plasmid was transformed into bacteria, induced and expressed by IPTG, the bacteria were disrupted, and 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 method according to claim 1, characterized in that: in step (e), the primer sequences used in PCR enrichment of the template are the Read1 and Read2 sequences common to the Illumina sequencing platform, and the Read1 sequence is as shown in SEQ ID NO: 21 As shown, the Read2 sequence is shown in SEQ ID NO: 22. The method according to claim 1, characterized in that: in step (e), 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 magnetic beads and the enriched template are purified at a ratio of 1:1. The method according to claim 1, wherein in step (f), one P5 and one P7 form a pair of primers, and the primers are adapted to the Illumina sequencing platform for sequencing, and the primer sequences of P5 and P7 are as follows: P5:AATGATACGGCGACCACCGAGATCTACAC[Index]TCGTCGGCAGCGTC, P7: CAAGCAGAAGACGGCATACGAGAT[Index]GTCTCGTGGGCTCGG, Where Index is a nucleic acid sequence of 8 bp in length. The application of the method for constructing a single-cell transcriptome sequencing library according to any one of claims 1-7 in single-cell sequencing. A single-cell sequencing method adopts the single-cell transcriptome sequencing library construction method of any one of claims 1-7 to construct a single-cell sequencing library, and uses a high-throughput sequencing platform for sequencing. The method for constructing a single-cell transcriptome sequencing library according to any one of claims 1-7 or the single-cell sequencing method according to claim 9 is used in the preparation of detection kits, detection devices or detection systems for developmental research or cancer research Applications.

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