CN110684829A - High-throughput single-cell transcriptome sequencing method and kit - Google Patents
High-throughput single-cell transcriptome sequencing method and kit Download PDFInfo
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Abstract
The application discloses a high-throughput single-cell transcriptome sequencing method and a kit. The high-throughput single-cell transcriptome sequencing method comprises the steps of wrapping single cells and microbeads with labels in a droplet by adopting a droplet generation system, and carrying out reverse transcription in the droplet. According to the method, the flux can reach 9000 cells, which is equivalent to 10 × genomics, the mutual pollution of the microbeads is small after the liquid drops are demulsified, and the effective data ratio is improved. The method carries out reverse transcription in the liquid drop, and has the advantages of less required reagent and low cost. In a preferred scheme of the application, the reverse transcription adopts a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; avoids the deviation caused by multiple amplifications, realizes the connection of the droplet microfluidic platform and the BGISeq-500 sequencing platform, simplifies and facilitates the large-scale single-cell sequencing.
Description
Technical Field
The application relates to the field of single cell sequencing, in particular to a high-throughput single cell transcriptome sequencing method and a kit.
Background
The disease occurrence mechanism and their detection and prevention are mainly analyzed by detecting the variation of cellular genome and abnormal expression of transcriptome. The single cell sequencing technology can reveal the complex heterogeneity of cells in tissues, and provide accurate information for diagnosis and treatment of diseases. Due to the problems of processing flux, cost and the like of the single cell sequencing technology, a lot of large-scale research work cannot be carried out, and the problems can be well solved by combining the microfluidic technology and the single cell sequencing technology; meanwhile, the single-molecule sequencing technology can avoid errors caused by nucleic acid amplification, and has great application prospect and requirements in basic scientific research and clinical medical research.
The instrument throughput has been increased to a point where hundreds of single cells can be analyzed at one time with a fully automated system of C1 single cells, introduced by Fludigm corporation in 2014. The advent of this system has been the focus of attention of the world-wide group of single-cell research topics. However, the Fluidigm C1 system, which uses micro valves to distinguish single cells, can only do 938 cells at most due to the limited chambers on the chip and low throughput, and is costly.
The Wafergen system uses a micropore chip and a mechanical arm to realize the separation of single cells, the number of micropores is 72, and compared with the Fluidigm C1 system, the Wafergen system flux can reach 1000-; however, the flux is still low, and the use requirement of large-scale research work cannot be well met; in addition, the wafer system has a large reaction system, so that a large amount of reagents are consumed, the cost is high, and the sample loss amount is large.
Compared with a Fludigm C1 system and a Wafergen system, the 10 Xgenomics and Dolomite flux based on the droplet microfluidic principle is higher, microbeads with labels and single cells are wrapped in one droplet by utilizing a microfluidic chip, the single cells are separated and marked, and tens of thousands of cells can be analyzed simultaneously. However, when the Dolomite system breaks emulsion, the no-load primers on the microbeads are easy to combine with free mRNA, so that the cross contamination is large; the formed library cannot be directly cyclized and cannot be directly connected with a BGISeq-500 platform; additional library transformations are required, introducing data bias, and reducing the effective utilization rate of data. The 10 Xgenomics has high requirements on samples and high cost; the formed library can not be directly cyclized, and can not be directly connected with a BGISeq-500 platform; likewise, additional library transformations are required, introducing data bias, reducing the effective use of data.
In addition, there are several single cell separation techniques, such as oral pipettes, flow cytometry, etc. However, the mouth pipette is micro-operated, the method has low flux, long time consumption and complicated process. The flow cytometer has the advantages of large sample demand, accurate control, damage to cells and high requirement on subsequent library construction.
In general, the existing single cell isolation or single cell transcriptome sequencing technology is limited by the problems of flux, cost and the like, and a lot of large-scale research work cannot be carried out. On the one hand, low throughput is more difficult to achieve in detail analysis of cellular heterogeneity and different cell subsets. On the other hand, the increase of the double-packet rate caused by the increase of the flux becomes a bottleneck of the current high-flux platform.
Disclosure of Invention
It is an object of the present application to provide a novel high throughput single cell transcriptome sequencing method and kit.
The application specifically adopts the following technical scheme:
the first aspect of the application discloses a high-throughput single-cell transcriptome sequencing method, which comprises the steps of wrapping single cells and microbeads with labels in one droplet by using a droplet generation system, and carrying out reverse transcription in the droplet to obtain first-strand cDNA.
It should be noted that, the single-cell transcriptome sequencing method of the present application, which adopts a droplet technology to separate single cells, has a throughput of about 9000 cells in a single experiment, which is equivalent to the highest throughput of 10 × genomics in the current market. The method realizes and provides reverse transcription in the liquid drop through research, and has the advantages that firstly, mutual pollution of microbeads after the liquid drop demulsification is effectively reduced, and the ratio of effective data is improved; secondly, reverse transcription is carried out in the liquid drops, and the required reagent is relatively less, so that the cost is low; lays a foundation for large-scale single cell research work.
Preferably, reverse transcription employs SMART template switching technology to introduce bases at the 5 'end of the first strand cDNA of the transcript of mRNA to distinguish from 3' end sequences.
The single-cell transcriptome sequencing method obtains the first-strand cDNA by a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; the method not only avoids the deviation caused by multiple amplifications in the library transformation process, but also realizes the combination of the droplet microfluidic platform and the BGISeq-500 sequencing platform, greatly simplifies and facilitates large-scale single cell sequencing.
Preferably, the single-cell transcriptome sequencing method further comprises performing SMARTPCR pre-amplification on the SMART template conversion product by using SMART PCR PRIMER, and obtaining a 3' end marker sequence by amplification; wherein SMARTPCRPRIMER is a specific primer at the 3' end.
It should be noted that the purpose of SMARTPCR pre-amplification is to amplify a target product at the 3 'end, so as to facilitate specific amplification of a labeled 3' sequence in the subsequent library construction. In one implementation of the present application, a specific tag, i.e., the sequence shown in SEQ ID NO.5, is added to the 3' end at the same time as the reverse transcription of the switch template.
Preferably, SMARTPCRPRIMER is the sequence shown in SEQ ID NO. 1;
SEQ ID NO.1：5’-AAGCAGTGGTATCAACGCAGAGT-3’。
it should be noted that SMARTPCRPRIMER of the sequence shown in SEQ ID NO.1 is only a specific implementation manner in the examples of the present application, and it is understood that other 3' end specific primers can be used under the basic concept of the present application, and are not limited herein.
Preferably, the single-cell transcriptome sequencing method further comprises the steps of performing PCR amplification enrichment on the 3 'end marker sequence by using a specific primer group aiming at the 3' end marker sequence, then performing fragment selection on the PCR amplification enrichment product, and directly sequencing by using a BGISeq-500 platform after cyclizing the selected fragment.
It should be noted that PCR amplification enrichment is a step in the construction of Tn5 library, and after PCR amplification enrichment and fragment selection, a more accurate sequencing library can be obtained, thereby improving sequencing efficiency and quality.
Preferably, the specific primer group adopted in the PCR amplification enrichment has an upstream primer with a sequence shown by SEQ ID NO.2 and a downstream primer with a sequence shown by SEQ ID NO.3, and the 5' end of the upstream primer has phosphorylation modification;
SEQID NO.2：
5’-GAACGACATGGCTACGATCCGACTTAAGCAGTGGTATCAACGCAG AGTAC-3’
wherein, the last two bases of the upstream primer of the sequence shown in SEQ ID NO.2 are modified by sulfo;
SEQ ID NO.3：
5’-TGTGAGCCAAGGAGTTGTTGTCTTCGTCTCGTGGGCTCGG-3’；
the Splint oligo adopted for fragment cyclization is a sequence shown in SEQ ID NO.4,
SEQ ID NO.4：5’-GCCATGTCGTTCTGTGAGCCAAGG-3’。
it should be noted that the primers with sequences shown in SEQ ID nos. 2 and 3 and the Splint oligo with sequence shown in SEQ ID No.4 are also a specific implementation manner in the embodiment of the present application, and it is understood that other specific primer sets or Splint oligos can be adopted under the basic concept of the present application, and are not limited herein.
It should be noted that, in the downstream primer of the sequence shown in SEQ ID No.3, in order to distinguish samples from different sources, an index sequence of about 10bp may also be introduced into the primer sequence, for example, in an implementation manner of the present application, the downstream primer of the following sequence is specifically adopted:
5’-TGTGAGCCAAGGAGTTGTTGTCTTCNNNNNNNNNNGTCTCGTGG GCTCGG-3’。
preferably, in the reverse transcription, 1mL of the reverse transcription reaction solution comprises: 10 μ L of 10% Triton-X, 125 μ L, RnaseOUT 75 μ L of 0.1M DTT, 50 μ L of 10Mm each dNTPs, 400 μ L of 5 × RTbuffer, 75 μ L of Super Script IIReverse Transcriptase, 20 μ L, Rnase-free H of Template Switch Oligo2O265. mu.L; the reaction conditions of reverse transcription are 30min at 37 ℃, 60min at 65 ℃ and standby at 4 ℃.
It should be noted that the single-cell transcriptome sequencing method of the present application provides, in particular, a reverse transcription reaction system suitable for the present application, which has not been performed in vitro or in droplets in the prior art.
Preferably, the Template Switch Oligo has the sequence shown in SEQ ID NO.5,
SEQ ID NO.5：5’-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3’
in the Template Switch Oligo of the sequence shown in SEQ ID NO.5, the penultimate bases 2 and 3 are riboguanoside modified, and the last base is LNA modified.
It should be noted that a Template, namely a SMART conversion Template, and a Template, namely a Template of a SMART Switch shown in SEQ ID No.5, are also a specific implementation manner in the embodiments of the present application, and it is to be understood that, under the basic concept of the present application, other SMART conversion templates may also be used, and are not specifically limited herein.
Preferably, in the labeled microbeads, the label is a primer sequence with a poly T random sequence, the primer sequence is a sequence shown in SEQ ID NO.6, and the 5 'end and the 3' end of the primer sequence are respectively provided with a poly T tail; two sections of barcodes are arranged between the 3 'end and the polyT tail of the 3' end of the primer sequence, the first section of barcode is cellbarcode and is used for marking single cells, and the second section of barcode is used for marking transcripts;
SEQ ID NO.6：5’-AAGCAGTGGTATCAACGCAGAGTAC-3’。
it should be noted that, the primer sequence with poly T random sequence in the bead can hold mRNA released after cell lysis; wherein, the first section of barcode is cell barcode, is a random sequence, is generally represented by J12 and is used for marking single cells, and the cellbarcodes on the same microbead are the same; a second segment of barcode, also a random sequence, generally designated N8, also known as UMI, is used to label transcripts that differ in UMI on the same microbead.
Preferably, the tag sequence is a sequence shown in SEQ ID NO.7,
SEQ ID NO.7：
5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’
in the sequence shown in SEQ ID NO.7, the first 12 Ns are the random sequence of the first segment of barcode, and the last 8 Ns are the random sequence of the second segment of barcode.
It should be noted that the tag sequence of the sequence shown in SEQ ID No.7 is also a specific implementation manner in the embodiments of the present application, and it is understood that other tag sequences can also be used under the basic concept of the present application, and are not limited herein.
The other side of the application discloses a kit for sequencing a high-throughput single-cell transcriptome, which comprises a SMART conversion template, a cDNA 3' end marker primer, a PCR amplification enrichment primer group, a Splint oligo and a microbead with a label; wherein the SMART conversion template is a sequence shown in SEQ ID NO.5 and is used for introducing base at the 5 'end of the first strand cDNA of the transcript of mRNA so as to be distinguished from a 3' end sequence; the cDNA3 'end marker primer is a sequence shown in SEQ ID NO.1 and is used for obtaining a 3' end marker sequence in amplification; the upstream primer of the PCR amplification enrichment primer group is a sequence shown by SEQ ID NO.2, and the downstream primer is a sequence shown by SEQ ID NO.3, and is used for carrying out PCR amplification enrichment on a 3' end marker sequence; splint oligo is a sequence shown in SEQ ID NO.4 and is used for circularizing fragmented nucleic acid; in the microbeads with the labels, the sequence of the labels is shown in SEQ ID NO.7, wherein the first 12N are random sequences of a first section of barcode, and the last 8N are random sequences of a second section of barcode.
It should be noted that the kit of the present application is actually the reagents used in the high-throughput sequencing method for single-cell transcriptome in the embodiment of the present application, and the reagents are combined into the kit, so that the high-throughput sequencing for single-cell transcriptome can be conveniently performed. It is to be understood that the kit may further include a reaction solution, an enzyme, and the like required in each reaction system for convenience of use, and is not particularly limited herein.
The beneficial effect of this application lies in:
according to the high-throughput single-cell transcriptome sequencing method, the single experiment flux can reach about 9000 cells, which is equivalent to the highest flux of 10 × genomics in the current market, and the microbeads subjected to liquid drop demulsification have small mutual pollution, so that the ratio of effective data is increased; in addition, the method carries out reverse transcription in the liquid drop, and has relatively less required reagent and low cost; lays a foundation for large-scale single cell research work. In a preferred scheme of the application, the reverse transcription adopts a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; not only avoids the deviation caused by multiple amplifications, but also realizes the connection of the droplet microfluidic platform and the BGISeq-500 sequencing platform, greatly simplifies and facilitates large-scale single cell sequencing.
Drawings
FIG. 1 is an explanatory diagram of a case where a droplet generating system generates droplets in a chip in an embodiment of the present application;
FIG. 2 is a graph showing the observation results of 1 μ L of beads-coated droplets under a 4-fold mirror in the examples of the present application;
FIG. 3 is a graph showing the results of detecting a reverse transcription product produced in a droplet using 2100 in the example of the present application;
FIG. 4 shows the 2100 detection results of the fragment selection products of PCR amplification-enriched products after PCR amplification enrichment of the marker sequences at the 3' end by the specific primer set during the Tn5 library construction in the present example.
Detailed Description
High-throughput single-cell transcriptome sequencing is of great significance in basic scientific research and clinical medical research. Although high throughput single cell transcriptome sequencing protocols such as the C1 system, the Wafergen system, the 10 × genomics and Dolomite are available abroad, these protocols have some disadvantages; in addition, the high-throughput single cell sequencing technology is still blank in China at present. The application first establishes a sequencing platform of a high-throughput single-cell transcriptome. Combining droplet microfluidics and single cell RNA sequencing technology SMART-Seq, using labeled microbeads to capture mRNA, simultaneously performing reverse transcription experiments in droplets, performing high-throughput label separation and obtaining the 3 'ends of thousands of single cell mRNA, distinguishing the 3' end sequences and the 5 'end sequences of the mRNA by adding a plurality of basic groups on a TSO sequence, skillfully designing specific primers, and amplifying and enriching the products with the labeled 3' ends. The cost is reduced while the single cell flux is improved, the single experiment cell flux is 2000-9000, and the cost is reduced to 1-0.35 yuan per cell. Reverse transcription is carried out in liquid drops, so that the pollution rate is reduced and the operation is simplified; simplifies the library building process and realizes the direct use with a BGISeq-500 sequencer.
The cell flux is improved, the cost is reduced, the flux is equivalent to the highest 10 × genomics in the current market, about 9000 cells can be achieved in a single experiment, the reagent cost is one third of the reagent cost of 10 × genomics, and the labor cost is greatly reduced. Makes it possible to carry out single cell sequencing research on a large scale. Compared with the existing method, the method has the advantages that the reverse transcription is carried out in the liquid drop, the mutual pollution of the microbeads after the liquid drop demulsification is effectively reduced, and the ratio of effective data is improved. In addition, the application designs a library construction system and a sequencing scheme aiming at the BGISeq-500, compared with other commercial platforms, the library constructed by the application can be directly used for BGISeq-500 sequencing, and the data error caused by multiple PCR in library conversion is reduced.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
1. Preparation of reagents
(1) Cell resuspension
PBS-BSA5mL with BSA final concentration of 0.01% was prepared; the H1975 cell line was washed with PBS-BSA and the cells were resuspended in PBS-BSA for further use.
(2) Reagent preparation for cleavage and reverse transcription
1mL of lysis and reverse transcription reagents included: 10 μ L of 10% Triton-X, 125 μ L, RnaseOUT 75 μ L of 0.1M DTT, 50 μ L of 10Mm each dNTPs, 400 μ L of 5 XT buffer, 75 μ L of Super Script II reverse transcription, 20 μ L, Rnase-free H of Template Switch Oligo2O 265μL。
Wherein the Template Switch Oligo is a sequence shown in SEQ ID NO.5,
SEQ ID NO.5：5’-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3’
in the Template Switch Oligo, the penultimate 2 and 3 bases are riboguanosine modified and the last base is LNA modified.
(3) Resuspension of microbeads
The beads of this example have primer sequences with poly (T) random sequences attached to them that allow for the capture of mRNA released after cell lysis. The primer sequence is shown as SEQ ID NO.6, and the sequence is shown as SEQ ID NO. 6: 5'-AAGCAGTGGTATCAACGCAGAGTAC-3' are provided.
The whole tag sequence is shown as SEQ ID NO.7,
SEQ ID NO.7：
5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’
wherein, the first segment of barcode is cell barcode, namely the first 12N, is a random sequence, is generally represented by J12 and is used for marking single cells, and the cellbarcodes on the same microbead are the same; the second barcode, also a random sequence, the last 8N, generally designated N8, also known as UMI, is used to label transcripts that differ from UMI on the same bead.
Centrifuging a proper amount of microbeads at 1000g for 1min, then carrying out resuspension cleaning at least once by using a lysis solution without DTT, and then carrying out resuspension on the microbeads by using the lysis solution without DTT, wherein the final concentration of the resuspension is 300 microbeads/mu L.
Wherein, 1mL of lysate without DTT comprises 10% Triton-X10 μ L, RnaseOUT 75 μ L, 10Mmeach dNTPs 50 μ L, 5 × RTbuffer400 μ L, Super Script II Reverse Transcriptase75 μ L, Template Switch Oligo 20 μ L, Rnase-freeH2O 265μL；
2. Droplet generation
The droplets are generated using a conventional droplet generation system. Before generating the liquid drops, the air in the tube is firstly exhausted by 30mL/h, and then the speeds of the three flow passages are controlled as follows: the flow rate of the oil phase is 10mL/h, the flow rate of the microsphere phase is 4mL/h, and the flow rate of the cell phase is 4mL/h, so that liquid drops are generated.
The case of generating droplets in a chip by a droplet generation system is shown in fig. 1. The resulting droplets were collected in an amount of 1. mu.L and observed under a 4-fold microscope, and the results are shown in FIG. 2. The results in FIG. 2 show that uniform droplets of single cells and bead coatings were obtained in this example preparation.
3. Reverse transcription in droplets
The resulting droplets (200. mu.L) were added to 1mL of the lysis and reverse transcription reagents and mixed well. Then, the mixture is placed at 37 ℃ for reaction for 30min, at 65 ℃ for reaction for 60min, and stands by at 4 ℃. Wherein, each liquid drop comprises microspheres, cells and reagents for lysis and reverse transcription; thus, cleavage and reverse transcription reactions can be performed independently in each droplet. The addition of 200. mu.L droplets to 1mL of lysis and reverse transcription reagents was intended to provide a lysis and reverse transcription reaction environment on a macroscopic scale.
4. Demulsification
a) Removing the bottom excess oil after reverse transcription reaction using a 1mL gun;
b) 30mL of 6 XSSC at room temperature was added;
c) adding 1mL of PFO surfactant into a fume hood, and vigorously shaking;
d) centrifuging at 1000 Xg for 1 min;
e) carefully taking the centrifuge tube out of the centrifuge, putting the centrifuge tube into ice, and removing the supernatant by using a pipettor until a small amount of liquid remains;
f) adding 30mL of 6 XSSC into the solution, standing to enable most of the oil to sink to the bottom, transferring the supernatant into another 50mL centrifuge tube, and floating the microbeads in the supernatant;
g) centrifuging at 1000 Xg for 1 min;
h) the beads were left at the bottom and the supernatant carefully aspirated, leaving approximately 1mL of liquid; transferring the remaining 1mL of liquid mixture into a 1.5mL centrifuge tube by using a gun, and centrifuging to remove the supernatant;
i) the supernatant was removed by centrifugation after washing twice with 1mL of 6 XSSC.
5. Enzyme digestion
The purpose of the enzyme cleavage is to remove primers on the beads that do not bind mRNA, to prevent cross-contamination. The specific enzyme digestion reaction system comprises: 10 XExo I Buffer20 μ L, H2O170. mu.L, Exo I10. mu.L at 20U/uL.
Washing the product obtained by the step 4 and the step 4 with 1ml of 10mM Tris pH8.0, adding 200 mu L of enzyme digestion reaction system, and incubating at 37 ℃ for 45min while keeping oscillation; completing the enzyme digestion reaction.
Washing the enzyme digestion product once by using 1mL of TE-SDS, and washing twice by using 1mL of TE-TW; if the subsequent PCR is to be performed, 1mL of H is reused2Cleaning once; then 1mL of H2And (4) resuspending the solution.
6. PCR Pre-amplification
And (3) taking 20 mu L of enzyme digestion product, adding the enzyme digestion product into a counting plate, calculating the approximate concentration of the microbeads, adding the enzyme digestion product into the PCR tubes according to the amount of about 2000 microbeads added into each PCR tube, centrifuging, and adding 50 mu L of PCR reaction liquid. A50. mu.L PCR reaction solution contained: h2O24.60. mu.L, SMART PCR PRIMER 0.40.40. mu.L at 100. mu.M, and 25.00. mu.L of 2 XKapa HiFiHotstartReady mixture.
Wherein SMARTPCRPRIMER is a sequence shown in SEQ ID NO. 1;
SEQ ID NO.1：5’-AAGCAGTGGTATCAACGCAGAGT-3’。
the PCR reaction conditions were a hot lid temperature of 105 ℃ for 3min at 95 ℃ and then 6 cycles: 20s at 98 ℃, 45s at 65 ℃ and 3min at 72 ℃, and after 6 cycles, the following 11 cycles are carried out: 20s at 98 ℃, 20s at 67 ℃ and 3min at 72 ℃, 5min at 72 ℃ and Hold at 4 ℃ after 11 cycles.
7. cDNA purification and quality control
Each sample was selectively purified using 0.6 × AgencourtAmpur XP microbeads.
a) The PCR pre-amplification product was made up to 50. mu.L with sterile distilled water.
b) Vortex and shake the AMPure XP microbeads uniformly and suck 30 microliter of volume into 50 microliter of PCR pre-amplification product, gently blow the product by using a pipettor for 10 times and fully mix the product uniformly, and incubate the product at room temperature for 10 minutes.
c) The reaction tube was centrifuged briefly and placed in a magnetic rack to separate the beads from the liquid, and the supernatant was carefully removed after the solution cleared.
d) Keeping the EP tube in the magnetic rack all the time, add 200 μ Ι _ of freshly prepared 80% ethanol to rinse the magnetic beads, incubate for 30 seconds at room temperature and carefully remove the supernatant. The above steps were repeated for a total of two rinses.
e) The EP tube was kept in the magnetic rack all the time, and the beads were air dried for 3 minutes by uncapping.
f) Taking the EP tube out of the magnetic frame, and adding 10 mu L of sterilized ultrapure water for elution; performing vortex oscillation or lightly blowing and beating by using a pipettor to fully mix the components; centrifuging the reaction tube for a short time and placing the reaction tube in a magnetic rack to separate magnetic beads and liquid; after the solution was clear, the supernatant was carefully pipetted into a sterile EP tube and stored at-20 ℃.
The enzyme digestion product was detected by using a 2100 detection system, and the results are shown in FIG. 3. The results in FIG. 3 show that the curve is unimodal and has no small fragment tailing, indicating that the reverse transcription is successful, the fragment distribution of the product is 600-10000bp, the main peak is 2000bp, and the size of the cDNA fragment is met.
8. Tn5 library construction
(1) Fragmentation process
Unfreezing 5 XTAG Buffer at room temperature, and reversing the mixture up and down and uniformly mixing the mixture for later use; NF buffer or 0.1% SDS was confirmed at room temperature, and the tube wall was flicked to confirm the presence or absence of precipitation. If there is sinkingThe precipitate was heated at 37 ℃ and vigorously shaken and mixed well to dissolve the precipitate. The fragmentation treatment reaction system comprises: mu.L of the digestion product, 5 XTAG Buffer4 mu L, FragmentEnzymeAdvanced mixed solution V5S 0.6.6 mu L, H2The total amount of O is 20 mu L. After the reaction solution was mixed well, it was incubated at 55 ℃ for 7min, 5. mu.L of 0.1% SDS was added thereto at room temperature for 5min to terminate the reaction, and the mixture was placed on ice.
(2) Enrichment by PCR amplification
In order to specifically amplify the labeled 3 'sequence and directly circularize the subsequent product, and avoid introducing bias by multiple amplifications, PCR amplification is carried out on the fragmented product by using a 5' phosphorylation modified specific primer Droplet-Ad 153-F-tag.
An upstream primer, namely Droplet-Ad153-F-tag, is a sequence shown in SEQ ID NO. 2; the downstream primer, namely Ad153-R-tag, can adopt a sequence shown in SEQ ID NO. 3; the 5' end of the upstream primer has phosphorylation modification, and the last two bases of the upstream primer are subjected to thio modification.
SEQ ID NO.2：
5’-GAACGACATGGCTACGATCCGACTTAAGCAGTGGTATCAACGCAG AGTAC-3’
SEQ ID NO.3：
5’-TGTGAGCCAAGGAGTTGTTGTCTTCGTCTCGTGGGCTCGG-3’。
In the embodiment, a random sequence of 10bp is added into the downstream primer of the sequence shown in SEQ ID NO.3 to distinguish different samples, so that the downstream primer Ad153-R-tag is specifically the following sequence:
5’-TGTGAGCCAAGGAGTTGTTGTCTTCNNNNNNNNNNGTCTCGTGG GCTCGG-3’。
the PCR reaction system comprises: 5 XKAPAFidelity Buffer 10. mu.L, 10mM each dNTPs 1.5. mu.L, 20. mu.M Droplet-Ad153-F-tag 2.5. mu.L, 20. mu.M Ad153-R-tag 2.5. mu.L, 1U/. mu.L of KAPAHiFiDNApolymerase, fragmentation product 5-12.5. mu.L, H2The content of O is filled to 50 mu L.
After the PCR reaction system is uniformly mixed, the PCR tube is placed in a PCR instrument, the following reaction procedures are set, the temperature of a hot cover is 105 ℃, the temperature of 72 ℃ is 5min, the temperature of 95 ℃ is 3min, and then 18 cycles are carried out: 20s at 98 ℃, 15s at 60 ℃ and 25s at 72 ℃, and 5min at 72 ℃ and Hold at 4 ℃ after circulation is finished.
(3) Amplification product fragment selection
Each sample was selectively purified using 0.6 × +0.2 × agencourt XP microbeads. The method comprises the following specific steps:
a) the PCR amplification product was made up to 50. mu.L with sterile distilled water.
b) Vortex and shake the AMPure XP microbeads uniformly and suck 30 microliter of PCR product to 50 microliter of PCR product, gently blow the PCR product by using a pipettor for 10 times, and fully mix the PCR product uniformly and incubate the PCR product for 10 minutes at room temperature.
c) The reaction tube was centrifuged briefly and placed in a magnetic stand to separate the beads from the liquid, after the solution was clarified, the supernatant was carefully transferred to a clean EP tube and the beads discarded.
d) Vortex and shake the AMPure XP microbeads uniformly and suck 10 microliter of volume to supernatant, gently blow the mixture 10 times by using a pipettor, and fully mix the mixture, and incubate the mixture for 5 minutes at room temperature.
e) The reaction tube was centrifuged briefly and placed in a magnetic rack to separate the beads from the liquid, and the supernatant was carefully removed after the solution cleared.
f) Keeping the EP tube in the magnetic rack all the time, add 200 μ Ι _ of freshly prepared 80% ethanol to rinse the magnetic beads, incubate for 30 seconds at room temperature and carefully remove the supernatant.
g) The above steps were repeated for a total of two rinses.
h) The EP tube was kept in the magnetic rack all the time, and the beads were air dried for 3 minutes by uncapping.
i) Taking the EP tube out of the magnetic frame, adding 15 mu L of sterilized ultrapure water for elution, carrying out vortex oscillation or gently blowing and beating by using a pipette, fully and uniformly mixing, centrifuging the reaction tube for a short time, placing the reaction tube in the magnetic frame to separate magnetic beads and liquid, after the solution is clarified, carefully sucking the supernatant into the sterilized EP tube, and storing at-20 ℃.
The enzyme digestion product was detected by using a 2100 detection system, and the results are shown in FIG. 4. The results in FIG. 4 show that the curve is unimodal with a major peak of 498bp, corresponding to the desired product fragment size.
(4) Sample loading
a) Loading according to the total amount of the DNA mixed solution of 550ng, and determining the loading volume of each sample according to the sample loading base number;
b) in order to ensure the base balance during sequencing, the loading base number can only be multiple of 8 at present, and the loading base number is 8 XN when 1 lane is measured by 8 XN samples.
c) The conversion method comprises the following steps: for example, if 16 samples were pooled into 1 library, each sample would have a total amount of 550/16-34.375 ng, and the concentration of sample 1 would be 5 ng/. mu.L, then sample 1 would have a volume of 29.375ng/(5 ng/. mu.L) -6.875. mu.L; sample 2 was calculated according to this method.
d) The DNA mixture was frozen to-20 ℃ for further use.
(5) ssDNA cyclization
Circularizing the nucleic acid fragments in the DNA mixture as follows:
sample preparation: mu.L of 20. mu.M Splint oligo 5. mu. L, DNA mixture was added to make up to 70. mu.L without nucleic acid. After mixing uniformly, the mixture was placed on ice at 95 ℃ for 3min to obtain 70. mu.L of a mixed sample solution for use.
Wherein the Splint oligo is a sequence shown in SEQ ID NO.4,
SEQ ID NO.4：5’-GCCATGTCGTTCTGTGAGCCAAGG-3’。
the reaction system for ssDNA cyclization comprises: the sample solution was mixed in a total of 120. mu.L, 70. mu.L of 10 XTA buffer 12. mu.L, 100mM ATP or 100mM dATP 1.2. mu.L, 600U/. mu.L of T4DNaligase 0.42. mu.L, and no nucleic acid water 36.38. mu.L.
After the reaction solution is prepared, oscillating and throwing for 5 s; incubation at 37 ℃ for 1h, 4 ℃ hold, yielded the ligation-circularization product of ssDNA.
The concentration of dsDNA was measured by taking 1. mu.L of the product, and the result showed that the concentration of the obtained ssDNA ligation-cyclization product was 3.8 ng/. mu.L, which could be used for subsequent analysis.
(6) Enzyme digestion
a) The reaction was placed in an ice bath and added in sequence: 120. mu.L of ssDNA cyclization product, 10 × TAbuffer 0.8. mu.L, 20U/. mu.L of EXO I3.9. mu.L, 100U/. mu.L of EXO III 0.65. mu.L, 2.65. mu.L of nucleic acid-free acid, and a total of 128. mu.L.
After the reaction solution is prepared, oscillating and throwing for 5 s; incubating at 37 deg.C for 30min, and holding at 4 deg.C; obtaining the enzyme digestion product.
Taking 1 mu L of enzyme digestion product to measure the concentration of dsDNA; the results showed that the concentration of the cleaved product was 0.16 ng/. mu.L, which was used for subsequent analyses.
(7) Magnetic bead purification
a) Adding 170 mu L of PEG32 microbeads into the enzyme digestion reaction product, vortexing, shaking and mixing uniformly, and incubating for 10 minutes at room temperature;
b) centrifuging the reaction tube for a short time, placing the reaction tube in a magnetic rack to separate magnetic beads and liquid, and carefully removing supernatant after the solution is clarified;
c) keeping the EP tube in the magnetic frame all the time, adding 200 μ L of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30 seconds, and carefully removing the supernatant;
d) repeating the above steps, and rinsing twice in total;
e) keeping the EP tube in the magnetic frame all the time, opening the cover and drying the magnetic beads in air for 3 minutes;
f) taking out the EP tube from the magnetic frame, adding 40 μ L of sterilized ultrapure water for elution, performing vortex oscillation or gently blowing and beating by using a pipettor to fully mix uniformly, centrifuging the reaction tube for a short time, placing the reaction tube in the magnetic frame to separate magnetic beads and liquid, after the solution is clarified, carefully sucking the supernatant into the sterilized EP tube, and storing at-20 ℃; namely, a Tn5 library which can be directly used for a BGISeq-500 sequencing platform is obtained.
Taking 1 mu L of the purified product to measure the ssDNA concentration; the results show that the Tn5 library has a concentration of 1.64 ng/. mu.L, which can meet the sequencing requirements.
9. BGISeq-500 sequencing
Directly performing BGISeq-500 sequencing on the obtained Tn5 library, wherein:
a) one-strand sequencing primer
R1-cell-UMIbarcode:SEQ ID NO.8：
5’-ATCCGACTTAAGCAGTGGTATCAACGCAGAGTAC-3’
b) Double-strand sequencing primer
R2-insert:SEQ ID NO.9：
5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
R2-sample index:SEQ ID NO.10：
5’-TGTGAGCCAAGGAGTTGTTGTCTTC-3’
c)MDA-Splint oligo:SEQ ID NO.11：
5’-GACAACAACTCCTTGGCTCACAGAACGACATG-3’
d) Three-stage sequencing with PE 30+100+10
Meanwhile, the sequencing verification is carried out on the products of '6 and PCR pre-amplification' by adopting Sanger sequencing; the Tn5 library constructed in this example was verified by sequencing using Sanger sequencing.
10. Sequencing results
According to the quality inspection report of a BGISeq-500 sequencing platform, the qualified ESR of the Tn5 library constructed in the embodiment is more than 75%, and specifically, the ESR is 76.42%; q30> 80%, with Q30 specifically being 90.32.
The above results demonstrate that the method of reverse transcription in droplets of this example produces an acceptable sequencing library and can be used well with the BGISeq-500 sequencing platform.
In the example, Sanger sequencing is adopted to carry out sequencing verification on the products of '6 and PCR pre-amplification', and the sequencing result is a sequence shown in SEQ ID NO. 12; wherein, in the sequence shown in SEQ ID NO.12, the 1 st base to the 69 th base are sequence structures on the microbeads, and the sequencing result shows that the primers on the microbeads can successfully capture RNA; the 26 th base to the 37 th base are cellbarcode and are used for marking single cells; UMI from base 38 to base 45, used to label the transcript; the TSO sequence with the 5 'end from 1224 st base to 1247 th base at the 3' end of the sequence shown in SEQ ID NO.12 shows that the captured RNA is successfully subjected to reverse transcription in the liquid drop; the middle sequence of the sequence shown in SEQ ID NO.12, i.e., the 70 th to 1223 rd bases, is a cDNA sequence. Sanger sequencing verification showed that the method of this example was successful in capturing RNA and that the captured RNA was successfully reverse transcribed in the droplets.
Sanger sequencing is used for carrying out sequencing verification on the Tn5 library constructed in the embodiment, and the sequencing result is a sequence shown in SEQ ID NO. 13; in the sequence shown in SEQ ID NO.13, the 1 st base to the 103 th base are sequence structures on the microbeads; the bases from 67 th base to 78 th base are cell barcode for labeling single cells; the 79 th to 86 th bases are UMI for marking the transcript; the sequence from 317 th base to 363 th base of the 3' end of the sequence shown in SEQ ID NO.12 is ad153R-tag sequence, wherein the bases from 332 th base to 340 th base are sample index for labeling different samples; the middle sequence of the sequence shown in SEQ ID NO.12, i.e., the 104 th to 316 th bases, is a cDNA sequence. Sanger sequencing verification results show that the method successfully removes the intermediate products and the 5 'end products, specifically amplifies and enriches the 3' end target products.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.
SEQUENCE LISTING
<110> Shenzhen Huashengshengsciences institute
<120> high-flux single-cell transcriptome sequencing method and kit
<130>17I25785
<160>13
<170>PatentIn version 3.3
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<400>1
aagcagtggt atcaacgcag agt 23
<210>2
<211>50
<212>DNA
<213> Artificial sequence
<400>2
gaacgacatg gctacgatcc gacttaagca gtggtatcaa cgcagagtac 50
<210>3
<211>40
<212>DNA
<213> Artificial sequence
<400>3
tgtgagccaa ggagttgttg tcttcgtctc gtgggctcgg 40
<210>4
<211>24
<212>DNA
<213> Artificial sequence
<400>4
gccatgtcgt tctgtgagcc aagg 24
<210>5
<211>32
<212>DNA
<213> Artificial sequence
<400>5
aagcagtggt atcaacgcag agtgaatrgr gg 32
<210>6
<211>25
<212>DNA
<213> Artificial sequence
<400>6
aagcagtggt atcaacgcag agtac 25
<210>7
<211>82
<212>DNA
<213> Artificial sequence
<220>
<221>misc_feature
<222>(33)..(52)
<223>n is a, c, g, or t
<400>7
tttttttaag cagtggtatc aacgcagagt acnnnnnnnn nnnnnnnnnn nntttttttt 60
tttttttttt tttttttttt tt 82
<210>8
<211>34
<212>DNA
<213> Artificial sequence
<400>8
atccgactta agcagtggta tcaacgcaga gtac 34
<210>9
<211>34
<212>DNA
<213> Artificial sequence
<400>9
gtctcgtggg ctcggagatg tgtataagag acag 34
<210>10
<211>25
<212>DNA
<213> Artificial sequence
<400>10
tgtgagccaaggagttgttg tcttc 25
<210>11
<211>32
<212>DNA
<213> Artificial sequence
<400>11
gacaacaact ccttggctca cagaacgaca tg 32
<210>12
<211>1247
<212>DNA
<213> sequencing results of reverse transcription product Sanger
<400>12
aagcagtggt atcaacgcag agtacactag tgtgtaacca agcgcttttt tttttttttt 60
ttttttttta aagactgaat tctttatttg gaatgaaata ttcttgtctt acacagtaga 120
taataaaaag gaataacgta tacacattat taatcataaa tgaaaagaga aaaccagtgc 180
aaaatgcggc agacagtaca tctctaacat attgcaaagg ctgataccgg gacaacacta 240
cttcagaaag gtgccagcaa aatggtgaat gtgtgaaaac aaagaaaaat attgtgttta 300
tagggtgcag aaagtttccc agaaactgac agagcccatg catctctgca cccagaatac 360
acttagagaa taatttaacc atgacaatag ggactacaga aaatggtata ttgtgtataa 420
acctggcctc tctaatcgcc tccttatgtg cctggaacat cttgacgttg ttcatgttcg 480
actgccaata tttcacatat cctccgtggt ctgctgtcaa catccacatg tcattatgtg 540
accacgtcat ggccctcact gggctgtcgt gagcctgtaa tattgtttca aaattgaaag 600
tgagtccatt ccacagggta aactccccac tagaagctcc agtgaccaag cgtcttcctt 660
ctggagtcca cctaacaaca aatacaggac actttacttt atttgttgat gtccgaacaa 720
attttgttgt tactgcattc ataggattat tcaacattcc tataggtggg accagatcat 780
tgtaataacc tgcatcaggc tgaattgccc gcatatctct ctggtctctt tgccatattc 840
tgttctccaa atacttaatt acagatggat tgtagtctat ggtttttcgg ttcacagctt 900
ttctcattcg ttttccatca aaagtaagct gttgcattgc ttgctgttgt gcaaaatcag 960
gtcgcttata aaacagctgt cgaggtgcct ggtgctggaa ccttggcata tggaaaaaac 1020
gaggaggaga accaatttct gtagccatgg tgatgttttc cttctaggat acgtctggct 1080
ttgagcagag ctcctaaatg gccagcgaag cgttcgcctt cagagccaga aatgtctctc 1140
ttgtttggtc tccccacccc gatcctctca atcccgctcc tccaaaggag cagccgccat 1200
cttcccctat gggcccccca ttcactctgc gttgatacca ctgctta 1247
<210>13
<211>363
<212>DNA
<213> sequencing results of fragments screened after Tn5 disruption
<400>13
aatgatacgg cgaccaccga gatctacacg cctgtccgcg gaagcagtgg tatcaacgca 60
gagtaccgcc ctctccggac atggtgtttt tttttttttt tttggctctc tgctcctcct 120
gttcgacagt cagccgcatc ttcttttgcg tcgccagccg agccacatcg ctcagacacc 180
atggggaagg tgaaggtcgg agtcaacgga tttggtcgta ttgggcgcct ggtcaccagg 240
gctgctttta actctggtaa agtggatatt gttgccatca atgacccctt cattgacctg 300
tctcttatac acatctccga gcccacgaga ctaaggcgaa tctcgtatgc cgtcttctgc 360
ttg 363
Claims (10)
1. A high throughput single cell transcriptome sequencing method, characterized in that: comprises the steps of wrapping single cells and microbeads with labels in one liquid drop by using a liquid drop generating system, and carrying out reverse transcription in the liquid drop to obtain first strand cDNA.
2. The single cell transcriptome sequencing method of claim 1, characterized in that: the reverse transcription employs SMART template switching technology to introduce bases at the 5 'end of reverse transcribed first strand cDNA of mRNA to distinguish from 3' end sequences.
3. The single cell transcriptome sequencing method of claim 2, characterized in that: carrying out SMART PCR pre-amplification on the SMART template conversion product by adopting SMART PCRPRIMER, and obtaining a 3' end marker sequence by amplification; wherein SMARTPCR PRIMER is a specific primer at the 3' end.
4. The single cell transcriptome sequencing method of claim 3, wherein: the SMART PCR PRIMER is a sequence shown in SEQ ID NO. 1;
SEQ ID NO.1：5’-AAGCAGTGGTATCAACGCAGAGT-3’。
5. the single cell transcriptome sequencing method of claim 3, wherein: the method also comprises the steps of carrying out PCR amplification enrichment on the 3 'end marker sequence by adopting a specific primer group aiming at the 3' end marker sequence, then carrying out fragment selection on a PCR amplification enrichment product, cyclizing the selected fragment, and directly sequencing by adopting a BGISeq-500 platform.
6. The single cell transcriptome sequencing method of claim 5, wherein: the upstream primer of the specific primer group is a sequence shown by SEQ ID NO.2, the downstream primer is a sequence shown by SEQ ID NO.3, and the 5' end of the upstream primer has phosphorylation modification;
SEQ ID NO.2：5’-GAACGACATGGCTACGATCCGACTTAAGCAGTGGTATCAACGCAGAGTAC-3’
SEQ ID NO.3：5’-TGTGAGCCAAGGAGTTGTTGTCTTCGTCTCGTGGGCTCGG-3’
the Splint oligo adopted for fragment cyclization is a sequence shown in SEQ ID NO.4,
SEQ ID NO.4：5’-GCCATGTCGTTCTGTGAGCCAAGG-3’
wherein, the last two bases of the upstream primer of the sequence shown in SEQ ID NO.2 are modified by sulfo.
7. The single cell transcriptome sequencing method of any one of claims 1 to 6, wherein: in the Reverse transcription, 1mL of reaction solution comprises 10% Triton-X10. mu.L, 0.1M DTT 125. mu. L, RnaseOUT 75. mu.L, 10Mm eachDNTPS 50. mu.L, 5 XT buffer 400. mu.L, Super Script II Reverse Transcriptase 75. mu.L, Template Switch Oligo 20. mu. L, Rnase-free H2O265. mu.L; the reaction conditions of reverse transcription are 30min at 37 ℃, 60min at 65 ℃ and standby at 4 ℃.
8. The single cell transcriptome sequencing method of claim 7, wherein: the Template SwitchOligo has a sequence shown in SEQ ID NO.5,
SEQ ID NO.5：5’-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3’
in the Template Switch Oligo of the sequence shown in SEQ ID NO.5, the penultimate bases 2 and 3 are riboguanoside modified, and the last base is LNA modified.
9. The single cell transcriptome sequencing method of any one of claims 1 to 6, wherein: in the microbeads with the labels, the labels are primer sequences with poly T random sequences, the primer sequences are sequences shown in SEQ ID NO.6, and the 5 'end and the 3' end of the primer sequences are respectively provided with poly T tails; two sections of barcode are arranged between the 3 'end of the primer sequence and the poly T tail of the 3' end, the first section of barcode is cell barcode and is used for marking single cells, and the second section of barcode is used for marking transcripts;
SEQ ID NO.6：5’-AAGCAGTGGTATCAACGCAGAGTAC-3’；
preferably, the tag sequence is a sequence shown in SEQ ID NO.7,
SEQ ID NO.7：5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’
in the sequence shown in SEQ ID NO.7, the first 12 Ns are the random sequence of the first segment of barcode, and the last 8 Ns are the random sequence of the second segment of barcode.
10. A kit for high throughput single cell transcriptome sequencing, characterized in that: comprises a SMART conversion template, a cDNA 3' end marking primer, a PCR amplification enrichment primer group, a Splint oligo and a micro-bead with a label;
the SMART conversion template is a sequence shown in SEQ ID NO.5 and is used for introducing base at the 5 'end of the first strand cDNA of the transcript of mRNA so as to be distinguished from a 3' end sequence;
the cDNA3 'end marker primer is a sequence shown in SEQ ID NO.1 and is used for obtaining a 3' end marker sequence in amplification;
the upstream primer of the PCR amplification enrichment primer group is a sequence shown by SEQ ID NO.2, and the downstream primer is a sequence shown by SEQ ID NO.3, and is used for carrying out PCR amplification enrichment on the 3' end marker sequence;
the Splint olig is a sequence shown as SEQ ID NO.4 and is used for cyclizing fragmented nucleic acid;
in the microbead with the tag, the tag sequence is shown in SEQ ID NO.7, wherein the first 12N are random sequences of a first section of barcode, and the last 8N are random sequences of a second section of barcode.
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