CN113444770B - Construction method and application of single-cell transcriptome sequencing library - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly discloses a construction method and application of a single-cell transcriptome sequencing library. The method comprises the following steps: lysing the sorted single cells in an orifice plate, and performing reverse transcription by using a reverse transcription primer to synthesize first strand cDNA; synthesizing a second strand cDNA using a substitution synthesis reaction; fragmenting the double-stranded cDNA using Tn5 transposase; enriching the fragmented templates by PCR; and (3) labeling the enriched fragments by using Index PCR, and purifying to obtain a single cell transcriptome sequencing library. The invention directly carries out enzyme digestion on double-stranded cDNA by using Tn5 transposase, does not need to carry out PCR pre-amplification on cDNA, can reduce library preference and obtain more accurate transcriptome information; meanwhile, tn5 transposase is used, so that the library construction efficiency can be improved, and the library construction time can be shortened. The method has simple library construction steps, can detect more genes, effectively reduces the proportion of the low-abundance genes dropout, reduces the library construction time and cost, and is favorable for finer research on a single cell level.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to the field of single-cell sequencing, and particularly relates to a construction method and application of a single-cell transcriptome sequencing library.
Background
In the last decade, RNA sequencing (RNA-seq) technology has been rapidly developed, and has become an indispensable tool for research such as differential gene expression, mRNA splicing analysis and the like, and has been widely applied in the biomedical field. The existing RNA-seq methods are mostly based on cell populations, the results of which reflect only the average expression level of genes in multicellular populations and cannot exhibit differences in gene expression in each cell. The recently developed single-cell RNA-seq is a novel technology for sequencing RNA at the single cell level, can effectively solve the problems of cell heterogeneity of a tissue sample and transcriptome heterogeneity of cell populations covered by conventional RNA-seq, provides a novel research direction for analyzing the behavior, mechanism and relation with organisms of single cells, and becomes a novel approach for single-cell research.
At present, a single-cell RNA library construction method generally needs to pre-amplify a small amount of RNA in a single cell, such as reverse transcription of the RNA into cDNA, and then PCR amplification is carried out on two ends of the cDNA by adding known sequences, or in vitro transcription and amplification are carried out on the cDNA by using an IVT technology, but the two library construction modes cannot avoid the problems of uneven transcriptome coverage, large background noise, inaccurate quantification and the like caused by deviation introduced by PCR amplification; also, these methods can cause dropout, especially for genes with low and medium expression levels, due to loss of some transcripts during amplification and capture, which is important for analysis of gene-to-gene co-expression and contribution of low and medium expression genes to biological pathways. Most single cell transcriptome sequencing methods such as SCRB-Seq, drop-Seq, etc., although detecting a larger number of cells, only sequencing the 3' end of the transcript, with each cell detecting a smaller number of genes; the Smart-Seq method can amplify full-length cDNA by using template conversion, so that the gene detection quantity is increased, but the quantity of detected cells is small, the steps are complex, and compared with other methods, the cost is higher. In addition, the traditional ultrasonic and enzyme digestion fragmentation library construction method needs to realize library construction through complex steps such as terminal repair, splicing and the like. In order to be able to obtain efficiently accurate transcriptome characteristics of single cells, it is necessary to develop a new single cell transcriptome sequencing technique.
Disclosure of Invention
Aiming at the problems, the invention provides a construction method of a single-cell transcriptome sequencing library and application thereof, which can quickly and effectively construct a single-cell transcriptome, and the invention uses Tn5 transposase to directly carry out DNA fragmentation and connection reaction on double-stranded cDNA, does not need to carry out PCR pre-amplification on the cDNA, and can quickly and efficiently construct the single-cell transcriptome sequencing library. The method can detect more genes, effectively reduce the proportion of the low-abundance genes dropout, obtain more accurate transcriptome information, and reduce the time and cost of library establishment.
To achieve the above and other related objects, a first aspect of the present invention provides a method for constructing a single cell transcriptome sequencing library, comprising the steps of:
(a) Lysing the sorted single cells in an orifice plate;
(b) Reverse transcription is carried out by utilizing a reverse transcription primer to synthesize a first strand eDNA;
(c) Synthesizing a second strand cDNA using a substitution synthesis reaction;
(d) Fragmenting the double-stranded cDNA template with Tn5 transposase;
(e) Enriching the fragmented templates by PCR;
(f) The enrichment fragments are labeled by Index PCR, and a single cell transcriptome sequencing library which can be directly used for sequencing is obtained after the product is purified.
Further, in step (a), the single cells are cultured cells in vitro, tissue cells, and free cells in body fluids; the single cell is derived from one of any tissues of human beings, rats and mice; the single cells were sorted to lysate by flow cytometry and lysed on ice for 15 minutes to fully lyse the cells.
Further, in the step (b), the reverse transcription primer is an immobilization structure of TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN in order of 5 'to 3', which is used for specific binding to poly (A) tail of mRNA to ensure specificity of reverse transcription. Regarding the specific sequence and length range of the above-mentioned reverse transcription primer, those skilled in the art can design and use 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 such as t III reverse transcriptase or Superscript IV reverse transcriptase.
Further, in step (c), the second strand cDNA synthesis reagent used was NEBNext Ultra II Non-Directional RNA Second Strand Module kit from New England Biolabs.
Further, in the step (d), the Tn5 transposase is an In-House Tn5 transposase, and the preparation method of the In-House Tn5 transposase comprises the following steps: transforming bacteria with Tn5 recombinant plasmid, performing IPTG induced expression, crushing thalli, and purifying Tn5 transposase protein by using a Ni column; and transferring the assembly into a protein ultrafiltration concentration tube for ultrafiltration purification to obtain the Tn5 transposase. The In-House Tn5 transposase obtained by the method has the characteristics of good purity and high activity, and has high library construction efficiency.
Further, in the step (e), primer sequences used in the process of enriching the template by PCR are Read1 and Read2 sequences which are universal to an Illumina sequencing platform, and the Read1 sequence is shown as SEQ ID NO:21, the Read2 sequence is shown in SEQ ID NO: shown at 22.
Further, in step (e), the enriched template is purified by magnetic beads and then used for subsequent Index PCR, wherein the magnetic beads can be VAHTS DNA purification magnetic beads of Vazyme company or magnetic beads of the same type, and the magnetic beads and the enriched template are purified according to the ratio of 1:1.
Further, in step (f), one P5 and one P7 form a pair of primers that are adapted for Illumina sequencing platform sequencing, the primer sequences of P5 and P7 are as follows:
P5：AATGATACGGCGACCACCGAGATCTACAC[Index]TCGTCGGCAGCGTC，
P7：CAAGCAGAAGACGGCATACGAGAT[Index]GTCTCGTGGGCTCGG，
wherein Index is a nucleic acid sequence of 8bp in length. With respect to the specific sequence of Index, one skilled in the art can design a wider variety of classes for use as desired.
The second aspect of the invention provides application of the construction method of the single-cell transcriptome sequencing library in single-cell sequencing.
The third aspect of the invention provides a single-cell sequencing method, which comprises the steps of constructing a single-cell sequencing library by adopting the construction method of the single-cell transcriptome sequencing library, and then sequencing by adopting a high-throughput sequencing platform. Wherein, the high throughput sequencing platform is an Illumina sequencing platform.
The fourth aspect of the present invention provides a method for constructing the single-cell transcriptome sequencing library or the application of the single-cell sequencing method in preparing a detection kit, a detection device or a detection system for development research or cancer research.
As described above, the method for constructing the single cell transcriptome and the application thereof have the following beneficial effects:
compared with the existing Single cell sequencing technology, the scSTAT-seq (Single-cell Streamlined Transcription And Tagmentation Sequencing) technology directly performs enzyme digestion on double-stranded cDNA by using Tn5 transposase, does not need to perform PCR pre-amplification on a cDNA template, can effectively reduce library preference, and obtains more accurate transcriptome information; the In-House Tn5 transposase is used for synchronously completing DNA breaking and joint connection, so that the initial template quantity and sample processing time can be reduced, complex experimental operation is simplified, the library construction efficiency is effectively improved, and the library construction cost is reduced; the method has simple library construction steps, can detect more genes, effectively reduces the proportion of the low-abundance genes dropout, truly reflects the single-cell gene expression condition, is favorable for finer research on a single-cell level, and meets the requirement of single-cell sequencing development.
Drawings
FIG. 1 is a flowchart of the database creation according to an embodiment of the present invention.
FIG. 2 shows a library fragment profile of an embodiment of the invention.
FIG. 3 shows a graph of gene quantity distribution violin comparing the SMART-seq2 method according to the embodiment of the present invention.
FIG. 4 shows a sequencing saturation plot comparing the SMART-seq2 method of an embodiment of the present invention.
FIG. 5 shows a plot of the gene dropout of the present invention compared to the SMART-seq2 method.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The library construction in the present invention was performed based on mouse monocyte macrophage leukemia cell RAW 264.7.
The main reagents, instruments and materials used in the present invention are shown in the following table 1.
TABLE 1
The specific implementation process is as follows:
in this embodiment, for example, single cell library construction of a 96-well plate is taken, for example, more single cells need to be subjected to library construction, and more Index PCR primers need to be designed and synthesized. The method for constructing the single cell transcriptome of the present invention is specifically described by the following steps:
1. lysing cells
1. The solution was formulated and dispensed into 96 wells according to the following composition:
1％CA630--------------------------------------------------0.6μl
rnase OUT ribonuclease inhibitor of 0.025. Mu.l
The method has the advantages that the method is convenient to use, and the device is easy to use
Deoxyribonucleoside triphosphate premix solution of 0.1 μl
The method is characterized in that the method comprises the following steps of
The method has the advantages that the method is convenient to use, and the device is suitable for being used for carrying out the process of the method
Wherein the reverse transcription primer is an immobilization structure of TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN in order of 5 'to 3', which is used for specific binding to poly (A) tail of mRNA to ensure specificity of reverse transcription.
2. Single cells were sorted into 96-well plates using flow cytometry by re-suspending RAW 264.7 cells into PBS solution.
3. The cells were lysed on ice for 15 minutes.
2. Reverse transcription
1. The solution was formulated and dispensed into 96 wells containing lysed cells, the solution composition being as follows:
the utility model discloses a self-priming syringe for preventing and treating the fracture
The first chain buffer of the super-long buffer is used for preventing the first chain buffer from being damaged
Rnase OUT ribonuclease inhibitor (40 units/ul) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -track) 0. - -2) 0.2. Mu.l)
SuperScript III reverse transcriptase (200 units/ul) - -, 0.2. Mu.l
The method has the advantages that the method is convenient to use, and the device is easy to use
The method has the advantages that the method is convenient to use, and the device is suitable for the users
2. Reverse transcription reaction was performed: 25℃for 10 minutes, 35℃for 10 minutes, 37℃for 10 minutes, 50℃for 60 minutes, 70℃for 15 minutes, 4 ℃.
3. Second strand cDNA Synthesis
1. A solution was prepared and dispensed into 96 wells containing reverse transcription product, the solution composition was as follows:
the method is characterized in that the method comprises the following steps of
The utility model discloses a new structure of the motor vehicle
The method has the advantages that the method is convenient to use, and the device is convenient to use
The method has the advantages that the method is convenient to use, and the device is suitable for being used for carrying out the process of the method
2. Second strand cDNA synthesis procedure was performed: the temperature was kept at 16℃for 60 minutes and 4 ℃.
4. Fragmentation of double-stranded cDNA template
1. A solution was prepared and dispensed into 96 wells containing double stranded cDNA, the solution composition was as follows:
the method is characterized in that the method comprises the following steps of
Tn5 transposase is 1 μl
The method is characterized in that the method comprises the following steps of
The method has the advantages that the method is convenient to use, and the device is easy to use
Wherein the Tn5 transposase is the In-House Tn5 transposase prepared In the laboratory, and the preparation method of the In-House Tn5 transposase is as follows: transforming bacteria with Tn5 recombinant plasmid, performing IPTG induced expression, crushing thalli, and purifying Tn5 transposase protein by using a Ni column; and transferring the assembly into a protein ultrafiltration concentration tube for ultrafiltration purification to obtain the Tn5 transposase with high activity and high purity.
2. And (3) performing a fragmentation procedure: the temperature is kept at 55 ℃ for 10 minutes and 10 ℃.
3. Immediately after completion of the fragmentation procedure, the reaction was stopped by adding 5. Mu.l of 0.2% SDS, and the mixture was blown and mixed with a pipette and left at room temperature for 5 minutes.
5. PCR enrichment of fragmented templates
1. A solution was prepared and dispensed into 96 wells of a buccal tablet segmentation template, the solution composition being as follows:
the method has the advantages that the method is convenient to use, and the device is convenient to use
The preparation method comprises the steps of
The method comprises the steps of (1) carrying out detection on the substances in the substrate, and (2) carrying out detection on the substances in the substrate
The method comprises the steps of (1) carrying out detection on the substances in the substances by using a detection device, and (2) carrying out detection on the substances in the substances by using a detection device
The method is characterized in that the method comprises the following steps of
The method has the advantages that the method is convenient to use, and the device is suitable for being used for carrying out the process of the method
2. Fragment PCR enrichment procedure was performed: 72℃for 3 minutes, 98℃for 30 seconds, 25 cycles (98℃for 15 seconds, 60℃for 30 seconds, 72℃for 3 minutes), 72℃for 5 minutes, 4℃hold.
3. Fragment purification: mu.l of VAHTS DNA purification beads were added to each well, and the mixture was stirred and stirred with a pipette and left at room temperature for 5 minutes. The supernatant was removed after the sample was placed on a magnet rack for minutes. Wash 2 times with 100 μl80% ethanol. Mu.l of nuclease-free water was added, and the mixture was blown and mixed with a pipette and left at room temperature for 5 minutes.
6. IndexPCR
1. A solution was prepared and dispensed into 96 wells containing magnetic bead purification templates, the solution composition was as follows:
KAPA Hot Start HiFi high Fidelity enzyme mixture-the utility model discloses a device for treating the skin of the patient
The method comprises the steps of (1) carrying out a process of preparing the composite material by using the composite material, and (2) carrying out a process of preparing the composite material
The method comprises the steps of (1) carrying out the process of detecting the presence of a metal catalyst, and (2) carrying out the process of detecting the presence of a metal catalyst
The method has the advantages that the method is convenient to use, and the device is suitable for being used for carrying out the process of the method
Wherein, one P5 and one P7 form a pair of primers, which are suitable for the sequencing of an Illumina sequencing platform, and 96 pairs of primers are used for Index labeling of 96-well cells.
The primer sequences for P5 and P7 are as follows:
P5：AATGATACGGCGACCACCGAGATCTACAC[Index]TCGTCGGCAGCGTC，
P7：CAAGCAGAAGACGGCATACGAGAT[Index]GTCTCGTGGGCTCGG。
where Index is a nucleic acid sequence of 8bp in length, those skilled in the art can design a wider variety of sequences for use as desired.
In the embodiment, the P5 and P7 primers are from an Illumina library building kit, the P5 primers comprise P501, P502, P503, P504, P505, P506, P507 and P508, and the sequences of the P501-P508 are shown as SEQ ID NO. 1-SEQ ID NO. 8; the P7 primer comprises P701, P702, P703, P704, P705, P706, P707, P708, P709, P710, P711 and P712, and the sequences of the P701-P712 are shown as SEQ ID NO. 9-SEQ ID NO. 20.
2. Index PCR procedure was performed: 95℃for 30 seconds, 15 cycles (98℃for 20 seconds, 60℃for 15 seconds, 72℃for 1 minute), 72℃for 5 minutes, 4℃hold.
3. Index PCR products in 96-well plates were aspirated and purified using QIAquick PCR purification kit to obtain a sequencing library compatible with the Illumina sequencing platform.
7. Sequencing the constructed library.
1. After the library purification was completed, the fragment distribution of the library was examined using Agilent Fragment Analyzer, and the results of the examination are shown in FIG. 2.
2. The library obtained above was sequenced using the Illumina X10 platform and compared to the SMART-seq2 library, and the sequencing results by bioinformatics analysis are shown in figures 3, 4, 5. The primer sequences used above are shown in Table 2.
TABLE 2 primer sequence listing
As can be seen from FIG. 3, an average of up to 9000 genes could be found in RAW 264.7 cells, an average of 3000 more than SMART-seq 2.
As can be seen from fig. 4, while the SMART-seq2 library (blue curve) is saturated at about 50 tens of thousands of reads/cell, the scSTAT-seq library (red curve) is saturated later; however, at the same depth, scSTAT-seq library detected about 2000 more genes on average than SMART-seq2 library.
From fig. 5, it can be seen that the scSTAT-seq library (blue curve) showed lower dropout levels 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 invention directly performs enzyme digestion on double-stranded cDNA by using Tn5 transposase without performing PCR pre-amplification on cDNA templates, thereby effectively reducing library preference and obtaining more accurate transcriptome information; the In-House Tn5 transposase is used for synchronously completing DNA breaking and joint connection, so that the initial template quantity and sample processing time can be reduced, complex experimental operation is simplified, the library construction efficiency is effectively improved, and the library construction cost is reduced; the method has simple library construction steps, can detect more genes, effectively reduces the proportion of the low-abundance genes dropout, truly reflects the single-cell gene expression condition, is favorable for finer research on a single-cell level, and meets the requirement of single-cell sequencing development.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
SEQUENCE LISTING
<110> Chinese people's university of Legend army medical university
<120> construction method of single cell transcriptome sequencing library and application thereof
<130> PCQLJ203246
<160> 22
<170> PatentIn version 3.5
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<400> 21
tcgtcggcag cgtcagatgt gtataagaga cag 33
<210> 22
<211> 34
<212> DNA
<213> Artificial
<220>
<223> Read2
<400> 22
gtctcgtggg ctcggagatg tgtataagag acag 34
Claims (5)
1. A method for constructing a single cell transcriptome sequencing library, comprising the steps of:
(a) Lysing the sorted single cells in an orifice plate;
(b) Reverse transcription is carried out by using a reverse transcription primer to synthesize first-strand cDNA, wherein the reverse transcription primer is an immobilization structure which is TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN in sequence from 5 'to 3', the immobilization structure is used for carrying out specific binding with poly A tail of mRNA so as to ensure the specificity of reverse transcription, and reverse transcriptase used in reverse transcription is selected from one of Superscript II reverse transcriptase, superscript III reverse transcriptase and Superscript IV reverse transcriptase;
(c) Synthesizing second strand cDNA by substitution synthesis reaction, wherein the second strand cDNA synthesis reagent is NEBNext Ultra II Non-Directional RNASecond Strand Module kit of New England Biolabs company;
(d) Fragmenting the double-stranded cDNA template with Tn5 transposase;
(e) The method comprises the steps of enriching a fragmented template by using PCR, wherein primer sequences used in the process of enriching the template by using PCR are Read1 and Read2 sequences which are universal for an Illumina sequencing platform, the Read1 sequence is shown as SEQ ID NO. 21, and the Read2 sequence is shown as SEQ ID NO. 22; the enriched template is purified by magnetic beads and then is used for subsequent Index PCR, the magnetic beads are VAHTSDNA purified magnetic beads of Vazyme company or magnetic beads of the same type, and the magnetic beads and the enriched template are purified according to the proportion of 1:1;
(f) The enriched fragments were labeled using Index PCR, with one P5 and one P7 constituting a pair of primers that were adapted to Illumina sequencing platform sequencing, the primer sequences for P5 and P7 were as follows:
P5：AATGATACGGCGACCACCGAGATCTACAC-Index-TCGTCGGCAGCGTC，
P7：CAAGCAGAAGACGGCATACGAGAT-Index-GTCTCGTGGGCTCGG，
wherein Index is a nucleic acid sequence of 8bp in length;
the single cell transcriptome sequencing library directly used for sequencing is obtained after the product is purified.
2. The method according to claim 1, characterized in that: in the step (d), the Tn5 transposase is an In-House Tn5 transposase, and the preparation method of the In-House Tn5 transposase comprises the following steps: transforming bacteria with Tn5 recombinant plasmid, performing IPTG induced expression, crushing thalli, and purifying Tn5 transposase protein by using a Ni column; and transferring the assembly into a protein ultrafiltration concentration tube for ultrafiltration purification to obtain the Tn5 transposase.
3. Use of the method of construction of a single cell transcriptome sequencing library according to any of claims 1-2 in single cell sequencing.
4. A single cell sequencing method, wherein the single cell sequencing library is constructed by the single cell transcriptome sequencing library construction method according to any one of claims 1-2, and sequencing is performed by a high throughput sequencing platform.
5. Use of the method of construction of a single cell transcriptome sequencing library according to any of claims 1-2 or the single cell sequencing method according to claim 4 for the preparation of a detection kit, detection device or detection system for developmental or cancer research.
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| CN114107459B (en) * | 2021-11-11 | 2022-12-06 | 浙江大学 | High-throughput single cell sequencing method based on oligonucleotide chain hybridization marker |
| CN114350748A (en) * | 2021-12-30 | 2022-04-15 | 厦门大学 | Rapid single-cell library building method |
| WO2023155135A1 (en) * | 2022-02-18 | 2023-08-24 | 深圳华大生命科学研究院 | Construction method and sequencing method for single-cell transcriptome sequencing library and test kit for preparing single-cell transcriptome library |
| CN115386622B (en) * | 2022-10-26 | 2023-10-27 | 北京寻因生物科技有限公司 | Library construction method of transcriptome library and application thereof |
| CN115386624B (en) * | 2022-10-26 | 2023-03-21 | 北京寻因生物科技有限公司 | Single cell complete sequence marking method and application thereof |
| CN116536394A (en) * | 2023-04-26 | 2023-08-04 | 青岛百创智能制造技术有限公司 | Construction method of marine organism single cell transcriptome library |
| CN117683866A (en) * | 2024-01-22 | 2024-03-12 | 湛江中心人民医院 | Method for detecting DNA in cells |
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