CN115198001A - Single cell complete sequence transcriptome library construction method and application - Google Patents
Single cell complete sequence transcriptome library construction method and application Download PDFInfo
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Abstract
The invention belongs to the field of gene sequencing, and particularly provides a method for constructing a single-cell full-sequence transcriptome library and application thereof, wherein the method comprises the following steps: providing target mrnas for a plurality of cells; reverse transcribing the target mrnas of the plurality of cells using a reverse transcription primer pair to obtain a plurality of cDNA fragments of the plurality of cells; connecting a plurality of cDNA fragments of a plurality of cells with a plurality of tag bodies in a one-to-one correspondence manner to obtain a plurality of labeled cDNA fragments of each cell; and enriching each marked cDNA fragment, and introducing a library label to obtain the single-cell complete sequence transcriptome library. The invention adopts a forward primer with a random sequence and a reverse primer with a fixed sequence to carry out reverse transcription on target mRNA to form a plurality of cDNA segment products with sticky ends, introduces a molecular tag at any position of a cDNA full-length sequence through the sticky ends, and simultaneously connects different cell tags on cDNA segments from different cell sources, thereby distinguishing different cells and realizing high-throughput single-cell full-sequence sequencing.
Description
Technical Field
The invention belongs to the field of gene sequencing, and particularly relates to a construction method and application of a single-cell full-sequence transcriptome library.
Background
The high-throughput single-cell RNA detection mainly comprises three realization modes, namely a water-in-oil-based droplet separation technology, a microplate-based beads labeling technology and a microfluidic control mode. Water-in-oil based droplet partitioning techniques are represented by the 10 Xgenomics, drop-Seq and inderop platforms. The technology wraps the barcode marked micro-bead and a single cell in a single oil drop through a microfluidic technology and cracks to release RNA containing a polyA tail; each gel microbead is coupled with an oligo dT nucleic acid sequence containing a cell label and a molecular label; the mRNA is bound to oligo dT nucleic acid molecules containing cellular and molecular tags and then labeled with different cellular tags by reverse transcription to cDNAs from different cellular sources and used for later pool building and sequencing analysis. The beads labeling technique based on the microplate is represented by BD Cytoseq, seqwell and microwell-seq. The technology naturally sinks cells to a micropore array with more than ten times of the number of the cells to ensure the single cell entry rate, and then adds microbeads marked by cell labels into micropores to capture mRNA after cell lysis; the mRNA is combined with a reverse transcription primer containing a cell label and a molecular label, and then the different cell labels are marked on cDNA from different cell sources through reverse transcription and are used for later mixed library construction and sequencing analysis.
The single-cell RNA library construction technology mentioned above can only realize the base sequence detection near the specific 3 'tail end or 5' start end of RNA, but can not effectively realize the analysis of the RNA full-length sequence at the high-throughput single-cell level.
Disclosure of Invention
In view of this, the present invention provides a method for constructing a single-cell full-sequence transcriptome library, so as to obtain a cDNA labeled at any position of the cDNA and construct a cDNA full-sequence library.
In order to achieve the above object, the present invention provides a method for constructing a single-cell complete-sequence transcriptome library, wherein the method for constructing a single-cell complete-sequence transcriptome library comprises:
providing target mrnas for a plurality of cells;
performing reverse transcription on the target mRNAs of the plurality of cells by using a reverse transcription primer pair to obtain a plurality of cDNA fragments of the plurality of cells, wherein a forward primer of the reverse transcription primer pair comprises a first sequence and a random sequence, a reverse primer of the reverse transcription primer pair comprises a second sequence and a fixed sequence, and the second sequence is complementary to the first sequence;
connecting a plurality of cDNA fragments of a plurality of cells with a plurality of tag bodies in a one-to-one correspondence manner to obtain a plurality of labeled cDNA fragments of each cell, wherein each tag body comprises a cell tag, a molecular tag and a connecting sequence, the connecting sequence is complementary with the fixed sequence, the molecular tags of any two tag bodies are different, the cell tags of the tag bodies connected with the cDNA fragments of the same cells are the same, and the cell tags of the tag bodies connected with the cDNA fragments of different cells are different;
and enriching each marked cDNA fragment, and introducing a library label to obtain the single-cell complete sequence transcriptome library.
Optionally, the step of providing target mRNA of a plurality of cells comprises:
providing a plurality of detection regions and a plurality of cells to be detected;
depositing a plurality of cells to be detected in a one-to-one correspondence manner in a plurality of detection areas;
and (3) enriching the target mRNA of the deposited cells to be detected by adopting a capture body.
Optionally, the detection regions are arranged on a detection region integrated body, the detection region integrated body is arranged at intervals, and each detection region is a detection hole with an inner diameter of 10-200 um.
Optionally, the step of providing target mRNA of a plurality of cells comprises:
after cell lysis is carried out by adopting cell lysis solution, target mRNA enrichment is carried out by adopting a capture body, wherein the capture body comprises a capture body and a capture sequence connected to the capture body.
Alternatively, the capture sequence is oligo- (dT) n N is between 10 and 100, and the capture sequence is closed with a closure.
Optionally, the step of linking a plurality of the cDNA fragments of a plurality of cells to a plurality of tag bodies in a one-to-one correspondence to obtain a plurality of labeled cDNA fragments of each cell comprises:
providing a plurality of functional microbeads containing the tag bodies, wherein each functional microbead comprises a tag body and a plurality of breakpoints connected with the tag body, the cell tags and the molecular tags of each tag body jointly form each marking section, the connecting sequence is connected to one end of each marking section, the other ends of the marking sections containing the same cell tags are correspondingly connected with the breakpoints of the same type of functional microbeads one by one, the cell tags of the tag bodies of any two types of the functional microbeads are different, and the tag body and the marking sections can be conditionally disconnected at the breakpoints;
mixing a plurality of said cDNA fragments of the same cell with one of said functional microbeads, and a different plurality of said cDNA fragments with a different one of said functional microbeads;
disconnecting the tag body from the marking segment;
and connecting the connecting sequence with the cDNA fragments to obtain a plurality of labeled cDNA fragments.
Optionally, the breaking point is a PClinker which can be broken under ultraviolet illumination conditions.
Optionally, the tag body further comprises a primer binding sequence; the step of obtaining the single-cell complete sequence transcriptome library after enriching each marked cDNA segment and introducing a library label comprises the following steps:
and hybridizing and extending the marked cDNA segments in sequence, adding an amplification primer combined with the primer binding sequence, and performing index amplification to obtain the single-cell full-sequence transcriptome library.
In the invention, a forward primer with a random sequence and a reverse primer with a fixed sequence are adopted to carry out reverse transcription on target mRNA to form a plurality of cDNA segment products with sticky ends, a molecular tag is introduced into any position of a cDNA full-length sequence through the sticky ends, and different cell tags are connected to cDNA segments from different cell sources, so that different cells are distinguished, and high-throughput single-cell full-sequence sequencing is realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the construction of a single-cell full-sequence transcriptome library according to the present invention;
FIG. 2 is a diagram of the structure of a single cDNA fragment according to the present invention;
FIG. 3 is a schematic diagram of a trapping body according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a tag body according to an embodiment of the invention;
FIG. 5 is a flow chart of the construction of a single-cell full-sequence transcriptome library according to example 1 of the present invention;
FIG. 6 is a graph showing the size of the library fragments obtained in example 1 of the present invention;
FIG. 7 is a test chart of nucleic acid sequences in example 1 of the present invention.
The implementation, functional features and advantages of the present invention will be further described with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be apparent that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments.
It should be noted that those whose specific conditions are not specified in the examples were performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between the various embodiments may be combined with each other, but must be based on the realization of the capability of a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
In view of the fact that sequencing of the full-length sequence of cDNA cannot be performed in the high-throughput single-cell transcriptome library in the prior art, the main reason is that the current cell tags can only be labeled at the 3 'end or the 5' end of the full-length cDNA.
The invention provides a method for constructing a single-cell complete-sequence transcriptome library, which comprises the following steps as shown in figure 1:
step S10, providing target mRNA of a plurality of cells;
step S20, carrying out reverse transcription on the target mRNAs of the cells by adopting a reverse transcription primer pair to obtain a plurality of cDNA fragments of the cells, wherein a forward primer of the reverse transcription primer pair comprises a first sequence and a random sequence, a reverse primer of the reverse transcription primer pair comprises a second sequence and a fixed sequence, and the second sequence is complementary with the first sequence;
step S30, connecting a plurality of cDNA fragments of a plurality of cells with a plurality of tag bodies in a one-to-one correspondence manner to obtain a plurality of labeled cDNA fragments of each cell, wherein each tag body comprises a cell tag, a molecular tag and a connecting sequence, the connecting sequence is complementary to the fixed sequence, the molecular tags of any two tag bodies are different, the cell tags of a plurality of tag bodies connected with a plurality of cDNA fragments of the same cell are the same, and the cell tags of a plurality of tag bodies connected with a plurality of cDNA fragments of different cells are different;
and S40, enriching each marked cDNA fragment, and introducing a library label to obtain the single cell complete sequence transcriptome library.
In the invention, a random sequence of a forward primer with a random sequence is adopted to fragment cDNA so as to obtain sequence information of the whole mRNA, then a sticky end is formed with a reverse primer fixed sequence as a joint, as shown in figure 2, a plurality of cDNA segment products with sticky ends are formed, in figure 2, 1 is a first sequence, 2 is a random sequence, 3 is a reverse transcription product, 4 is a second sequence, and 5 is a sticky end formed by a fixed sequence, the cDNA segment products with the sticky ends are connected with a tag body with molecular tags and cell tags, so that the molecular tags are introduced into any position of the cDNA, meanwhile, the cDNA segment products from different cells are introduced with different cell tags, so that different cells are distinguished, a complete sequence library of a plurality of single cells is obtained at one time, and the detection flux is improved.
Specifically, in the present invention, after the 3 'end of the first sequence is ligated with a Random sequence, the reverse transcription forward Primer may be formed, and a cohesive end may be formed at the 5' end of the Primer for subsequent ligation, and the Random sequence may be a Random combination dN (6-20) of four base nucleotides of 6-20 ATGC, such as NNNNNN (N represents any base nucleotide of ATCG), or, of course, several bases may be fixed in the Random sequence, and the Random combination of any base nucleotide of ATCG may be used in addition to the fixed portion, for example, random Primer-F: gcttgtcttttnnnnnnn, wherein nnnnnnnn is used as a random primer for initiating a reverse transcription reaction, GCTTGTGTCTTTT is a complementary pairing sequence, the 3 'end of the second sequence is connected with a fixed sequence, and the finally formed cDNA fragment has a sticky end at the 5' end, so that a cell tag and a molecular tag are connected to each cDNA fragment, such as:
random Primer-R: AAAAGACACAAGCGBAAAAAAAAAAAAAAA, AAAAGACAAGC in sequence is used for complementary pairing with GCTTGTGTCTTTT in Random Primer-F;
the sequence is as follows: aaaaaaaaaaaaaaa is a fixed sequence as a cohesive end that mediates ligation reactions, the corresponding ligation sequence being ployT.
The first sequence and said second sequence are random combinations of any three of the four bases. The length of the first and second sequences ranges from 17 to 50 nucleotides, preferably it may be between 17 and 25 nucleotides. The melting temperature (Tm) of a duplex formed by the complementarity between the first sequence and the second sequence is 40 to 80 ℃, and more preferably 55 to 70 ℃.
In some embodiments, the step of providing target mRNA of a plurality of cells comprises:
step S101: providing a plurality of detection regions and a plurality of cells to be detected;
step S102: depositing a plurality of cells to be detected in a one-to-one correspondence manner in a plurality of detection areas;
step S103: and (3) enriching the target mRNA of the deposited cells to be detected by adopting a capture body.
By constructing reverse transcription libraries of different cells in a dividing region, the efficiency is improved, and meanwhile, the pollution of different cells is avoided. Specifically, a plurality of detection regions are arranged on a detection region integrated body, the detection region integrated body is arranged at intervals, and the inner diameter of each detection region is a detection hole of 10-200 um. In the present invention, the different detection regions are integrated in a microwell chip, which may contain 100 to 10000000 microwells, and the diameter of each microwell of the same chip is the same, and may be between 10-200 um. The number of cells put into the micro-porous chip should not exceed 1/5 of the number of micro-pores of the chip, and the cells can be naturally settled into the pores by gravity. When in use, the single cells are all carried out in a single micropore, and the pollution of different cell RNAs is avoided.
In some embodiments, step S10 comprises: the target mRNA is enriched by using a capture body, as shown in fig. 3, the capture body comprises a capture body and a capture sequence connected to the capture body, in fig. 3, 1 is the capture body, and 2 is the capture sequence. Specifically, in the invention, the capture sequence is oligo- (dT) n, the number of n is between 10 and 100, and the capture sequence is closed by a closing body; the enrichment of mRNA from eukaryotic cells can be achieved by using oligo- (dT) to complement the unique polyA of eukaryotic cells, and the enclosure is selected from one or more of free-arm modification, dideoxynucleotide modification, inverted dT modification, thio modification, locked nucleotide modification and phosphorylation modification.
In some embodiments, step S103 comprises: after the cell lysis is carried out by adopting cell lysis solution, the capture body is used for enriching the target mRNA. Specifically, cells and capture bodies are sequentially added into the microporous chip, then cell lysis solution is adopted to lyse the cells to release mRNA, and the capture bodies enrich target mRNA. By using cell lysis solution to perform cell lysis, mRNA in cells can be completely released.
It should be noted that, on the premise that the capture sequence can be connected, the material selection of the capture body is not limited, and specifically, in the present invention, the capture body is made of magnetic beads, and can be coupled with the capture sequence by a coupling reaction.
In some embodiments, step S30 includes:
step S301, providing a plurality of functional microbeads containing the tag bodies, wherein each functional microbead comprises a tag body and a plurality of breakpoints connected with the tag body, the cell tags and the molecular tags of each tag body jointly form each tag section, the connecting sequence is connected to one end of each tag section, the other ends of the tag sections containing the same cell tags are correspondingly connected with the breakpoints of the functional microbeads of the same type one by one, the cell tags of the tag bodies of any two kinds of the functional microbeads are different, and the tag bodies and the tag sections can be conditionally disconnected at the breakpoints;
step S302, mixing a plurality of cDNA fragments of the same cell with one functional microbead, and mixing a plurality of different cDNA fragments with different functional microbeads;
step S303, disconnecting the label body and the mark section;
and S304, connecting the connecting sequence with the cDNA fragments to obtain a plurality of labeled cDNA fragments.
By introducing the tag body, a large amount of cell tags can be loaded, and the binding rate can be improved. Specifically, in the present invention, one tag body load 10 5 ～10 10 A marker segment. Further, the conditional disconnection refers to that the mark segment and the mark body are broken at a breakpoint under a certain condition, specifically, disulfide modification, dU modification, RNA base modification, dI modification, DSpacer modification, AP site modification, photodisruption PC linker and a restriction enzyme recognition sequence. Further, in the invention, the breakpoint is a PClinker which can be broken under the condition of ultraviolet illumination.
In some embodiments, the tag body further comprises a primer binding sequence; the step S40 includes:
and hybridizing and extending the marked cDNA fragments in sequence, adding an amplification primer combined with the primer binding sequence, and performing index amplification to obtain the single-cell full-sequence transcriptome library. Specifically, the hybridization and extension methods in the present invention are based on the existing hybridization and extension procedures, and refer to patent CN 114096678A. Further, in the present invention, the technology of Beijing Hongyin technology company is adopted
MM kitThe hybridization, extension and amplification by the reagent (1).
In some embodiments, the specific structure of the functional microbead is shown in fig. 4, and includes a tag body 1, and a functional sequence 2 loaded on the tag body, wherein each functional sequence includes a connector 201, a breakpoint 202, a primer binding sequence 203, a cell tag 204, a molecular tag 205, and a connecting sequence 206 connected in sequence, wherein the connector is connected to the tag body, and the molecular tag 205 on one functional microbead is different, but the cell tag 204 is the same. By integrating sequences with different functions in one functional sequence, a reaction integrated body can be obtained, so that the construction efficiency of the library is improved. For example: the functional sequence is designed as follows:
<xnotran> ACACTCTTTCCC/PClinker/TACACGACGCTCTTCCGATCTnnnnnnnnACTGGTGAnnnnnnnnGGTAGTGACAnnnnnnnnNNNNNNNNTTTTTTTTTTTTTTTTTT, , ACACTCTTTCCC beads ; </xnotran> The primer binding sequence for the post-ligation cDNA amplification used for TACACGACGCTCTTCCGATCT, nnnnnnnnnnnnACTGGTGAnnnnnnnnnnnnnnnggTAGTGACAnnnnnnnnnnnnnnnnnnnn cell tag, NNNNN molecular tag, TTTTTTTTTTTTTTTT, and polyA in cDNA, mediate ligation, wherein each of N and N is selected from any base in ATCG.
It should be noted that, on the premise that the tag body of the present invention can be connected in sequence, the material of the tag body is not limited, and the tag body can be made of any hard material or soft material, such as solid beads, semi-solid hydrogel beads, and the like.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1
This example provides a construction and sequencing analysis of a single-cell full-sequence transcriptome library of PBMCs, the specific operations are shown in fig. 5:
1. peripheral blood was drawn and fresh PBMC cells (peripheral blood mononuclear cells) were obtained, resuspended in PBS;
2. providing a microwell chip, firstly putting 3000 incubated PBMC cells into the microwell chip, wherein the microwell chip can contain 100 to 10000000 microwells, and the diameter of each microwell of the same chip is the same and can be between 10 and 200 um;
3. add oligo (dT) washed with 300ul PBS 25 Dynabeads (polymer magnetic beads) are put into the micro-porous plate, and the redundant magnetic beads are cleaned after the Dynabeads are magnetically attracted into the pores;
4. adding lysis solution carried by the kit to perform lysis for 2min so that the magnetic beads adsorb RNA;
5. use of
Washing the chip for 1 time by Wash Buffer A and 2 times by Wash Buffer B in the MM 3' single cell library construction kit according to the following steps
The specification of the MM 3' single cell library construction kit configures 400ul of the following reverse transcription system:
and 2. Mu.l of random primer at a concentration of 100. Mu.M, which is an annealing product of the following two sequences, was additionally added.
| Primer name | Sequence of |
| Random Primer-F | gcttgtgtcttttNNNNNN |
| Random Primer-R | AAAAGACACAAGCAAAAAAAAAAAAAAAAAA |
Adding 350 mu L of prepared reverse transcription reaction system into a micropore plate, sealing, and then placing under the following temperature conditions for reaction:
| step (ii) of | Temperature of | |
|
| 1 | At | 10min | |
| 2 | 37℃ | 30min |
After completion of the reverse transcription reaction, the reaction mixture was washed 3 times with PBS and then washed
Cell barcoded beads were added by the method in the MM kit instructions. The specific sequence of coupling on the cell barcode beads is shown as follows:
6. preparing a connection reaction system according to the following system, adding the connection reaction system into a microporous plate, carrying out cracking for 1min by using ultraviolet light, and then placing the microporous plate at 22 ℃ for reaction for 15min;
the beads were removed from the plate and denatured at high temperature, the supernatant was transferred to a new 1.5ml centrifuge tube and purified by adding 1.6 XDNAclear beads to obtain cell-tagged cDNA product.
7. Adding the Beijing seeker technology corporation to step 6
The reagent bufferC c in the MM kit was placed in a constant temperature metal bath and the following procedure was run:
| temperature of | Time | Rotational speed | |
| 95 degree | 5min | ||
| 37 | 5min | 1500rpm | |
| 25 degree | 15min | 1500rpm |
8. According to the following
The instruction of the MM 3' single cell library construction kit configures 150ul of the following reaction system:
| reagent | 150 μ L system |
| BufferD | 141μL |
| ENzymeC | 6μL |
| ENzymeD | 3μL |
Adding the prepared Mix into the product obtained in the step 7, and reacting on a constant-temperature mixing machine, wherein the setting is as follows:
| temperature of | Time | Rotational speed |
| 37 degree | 10min | 1500rpm |
| 53 degree | 30min | 1500rpm |
9. After completion of the extension reaction, 0.9 XDNA clean beads were purified using the DNA chips of the Kyoto Hongyi scientific Co., ltd
The primers (Universal Oligo, I5, I7) in the MM kit were subjected to product enrichment and index addition, and the size of the obtained library fragment is shown in FIG. 6, and the length thereof is mainly concentrated in the range of 200 bp-1500 bp.
Comparative example 1
According to the Kyoto technology company of Beijing
The PBMC cells (peripheral blood mononuclear cells) were subjected to library construction by the procedure described in the MM kit.
Test examples
The illumina NovaSeq 6000 was sequenced, and the position of the detected nucleic acid sequence in the RNA was analyzed. As shown in FIG. 7, the nucleic acid sequence (A) determined by this method was distributed more evenly over the full length of the RNA than the SeekOne MM 3' transcriptome library (B).
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (8)
1. A method for constructing a single-cell complete-sequence transcriptome library, wherein the method for constructing the single-cell complete-sequence transcriptome library comprises the following steps:
providing target mRNA of a plurality of cells;
performing reverse transcription on the target mRNAs of the plurality of cells by using a reverse transcription primer pair to obtain a plurality of cDNA fragments of the plurality of cells, wherein a forward primer of the reverse transcription primer pair comprises a first sequence and a random sequence, a reverse primer of the reverse transcription primer pair comprises a second sequence and a fixed sequence, and the second sequence is complementary to the first sequence;
connecting a plurality of cDNA fragments of a plurality of cells with a plurality of tag bodies in a one-to-one correspondence manner to obtain a plurality of labeled cDNA fragments of each cell, wherein each tag body comprises a cell tag, a molecular tag and a connecting sequence, the connecting sequence is complementary with the fixed sequence, the molecular tags of any two tag bodies are different, the cell tags of the tag bodies connected with the cDNA fragments of the same cells are the same, and the cell tags of the tag bodies connected with the cDNA fragments of different cells are different;
and enriching each marked cDNA segment, and introducing a library label to obtain the single-cell complete sequence transcriptome library.
2. The method of single cell full sequence transcriptome library construction of claim 1, wherein said step of providing target mrnas for a plurality of cells comprises:
providing a plurality of detection areas and a plurality of cells to be detected;
depositing a plurality of cells to be detected in a one-to-one correspondence manner in a plurality of detection areas;
and (3) enriching the target mRNA of the deposited cells to be detected by adopting a capture body.
3. The method for constructing the single-cell full-sequence transcriptome library of claim 2, wherein a plurality of said detection regions are disposed on a detection region integrated body, and are arranged at intervals on said detection region integrated body, and each of said detection regions is a detection hole with an inner diameter of 10-200 um.
4. The method of single cell full sequence transcriptome library construction of claim 1, wherein said step of providing target mrnas for a plurality of cells comprises:
after cell lysis is carried out by adopting cell lysis solution, target mRNA enrichment is carried out by adopting a capture body, wherein the capture body comprises a capture body and a capture sequence connected to the capture body.
5. The method of constructing a single cell full sequence transcriptome library of claim 4, wherein said capture sequence is oligo- (dT) n N is in the range of 10 to 100, and the capture sequence is blocked with a blocking body.
6. The method of constructing a single-cell full-sequence transcriptome library of claim 1, wherein said step of linking a plurality of said cDNA fragments of a plurality of cells to a plurality of tag bodies in a one-to-one correspondence to obtain a plurality of tagged cDNA fragments of each cell comprises:
providing a plurality of functional microbeads containing the tag bodies, wherein each functional microbead comprises a tag body and a plurality of breakpoints connected with the tag body, the cell tags and the molecular tags of the tag bodies jointly form each tag section, the connecting sequence is connected to one end of each tag section, the other ends of the tag sections containing the same cell tags are correspondingly connected with the breakpoints of the functional microbeads of the same type one by one, the cell tags of the tag bodies of any two functional microbeads are different, and the tag bodies and the tag sections can be conditionally disconnected at the breakpoints;
mixing a plurality of said cDNA fragments of the same cell with one of said functional microbeads, and a different plurality of said cDNA fragments with a different one of said functional microbeads;
disconnecting the tag body from the marking segment;
and connecting the connecting sequence with the cDNA fragments to obtain a plurality of labeled cDNA fragments.
7. The method for constructing a single-cell full-sequence transcriptome library of claim 6, wherein said breakpoint is a PClinker cleavable under ultraviolet light conditions.
8. The method of single cell full sequence transcriptome library construction of claim 1, wherein said tag body further comprises a primer binding sequence; the step of obtaining the single-cell complete sequence transcriptome library after enriching each marked cDNA segment and introducing a library label comprises the following steps:
and hybridizing and extending the marked cDNA fragments in sequence, adding an amplification primer combined with the primer binding sequence, and performing index amplification to obtain the single-cell full-sequence transcriptome library.
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