CN112176422B - Method for constructing RNA library - Google Patents
Method for constructing RNA library Download PDFInfo
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- CN112176422B CN112176422B CN202011120991.9A CN202011120991A CN112176422B CN 112176422 B CN112176422 B CN 112176422B CN 202011120991 A CN202011120991 A CN 202011120991A CN 112176422 B CN112176422 B CN 112176422B
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
Abstract
The invention provides a construction method of an RNA library. The construction method comprises the following steps: reverse transcription is carried out on the fragmented RNA by adopting a reverse transcription primer to obtain first strand cDNA, and the reverse transcription primer sequentially comprises from the 5 'end to the 3' end: known sequences and random sequences; performing single-chain linker connection on the first-chain cDNA by using cyclase to obtain a connection product; and carrying out PCR amplification on the ligation products to obtain an RNA library. The RNA library can be obtained by introducing a first sequencing primer sequence while synthesizing first-strand cDNA through reverse transcription of RNA, then directly introducing a second sequencing primer sequence through single-link joint connection of cyclase, and finally designing a proper amplification primer according to the first sequencing primer sequence and the second sequencing primer sequence to perform PCR amplification on a joint connection product. The method can directly realize chain specificity without synthesis of second chain cDNA or other digestion processes, and has the advantages of simple operation and labor-saving.
Description
Technical Field
The invention relates to the field of RNA sequencing library construction, in particular to a construction method of an RNA library.
Background
The transcriptome sequencing technology, RNA-seq, is an important tool for researching biological gene expression, and the technology is widely applied to the analysis of gene expression profiles of different species. The recent RNA-seq library construction kit mainly focuses on RNA strand specificity research, the library construction process needs to extract total RNA and enrich mRNA, synthesize first strand cDNA through reverse transcription, synthesize second strand cDNA by dUTP, then perform terminal filling of double-stranded cDNA, add adenine A base and perform joint connection, digest cDNA containing uracil U base and then complete library construction through PCR amplification.
The library building process in the prior art is more complicated:
(1) dUTP is added to synthesize double-chain cDNA, and the chain specificity is distinguished through uracil U base;
(2) Carrying out terminal filling repair on the cDNA and adding adenine A base to the 3' end of the cDNA to ensure that the 3' end of the cDNA double-chain contains A base suspension and further matches with thymine T base at the 3' end of the joint to realize TA connection to complete library construction;
(3) Two single-chain linkers are required to be designed and annealed to form a double chain, and contain thiosulphate bond modification to ensure that the T basic group of the linker does not fall off, so that effective linker connection can be carried out;
(4) Finally, the second strand of the cDNA containing uracil U bases is digested prior to PCR to achieve strand-specific library construction.
Therefore, there is a need for improvement of the existing RNA library construction method to provide a simple and rapid library construction method.
Disclosure of Invention
The invention mainly aims to provide a construction method of an RNA (initial amount is 100ng-1000 ng) library so as to solve the problem of complex library construction process in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for constructing an RNA library, the method comprising: and carrying out reverse transcription on the fragmented RNA by using a reverse transcription primer to obtain first strand cDNA, wherein the reverse transcription primer sequentially comprises from the 5 'end to the 3' end: known sequences and random sequences; adopting cyclase to carry out single-chain linker connection on the first-chain cDNA to obtain a connection product; and carrying out PCR amplification on the ligation product to obtain an RNA library.
Further, the reverse transcription primer is upstream of the known sequence, and further comprises a spacer arm.
Further, the random sequence is a random sequence of 4 to 8 bases, and more preferably a random sequence of 6 bases.
Further, the Spacer arm is Spacer C12, spacer C6, spacer C3, NH 2 And ddC.
Furthermore, the sequence of the reverse transcription primer is SEQ ID NO. 1.
Further, the cyclase is cyclase II or T4 RNA ligase 1.
Further, the 5 'end of the single linker has an adenylation modification, and the 3' end has a ddC modification; preferably, the sequence of the single-stranded linker is SEQ ID NO 2.
Further, performing PCR amplification on the ligation product by using an upstream amplification primer and a downstream amplification primer to obtain an RNA library, wherein the upstream amplification primer sequentially comprises from 5 'end to 3' end: the sequence of the upstream sequencing platform, the sequence of the first library tag and the sequence of the upstream sequencing primer, wherein the downstream amplification primer sequentially comprises from 5 'end to 3' end: the sequence of the downstream sequencing platform, the sequence of the second library tag and the sequence of the downstream sequencing primer, wherein the sequence of the downstream sequencing primer is the same as at least part of the sequence of the 5 'end of the reverse transcription primer, and the sequence of the upstream sequencing primer is the same as at least part of the sequence of the 5' end of the single-stranded linker; preferably, the first library tag sequence and the second library tag sequence are each 4 to 10bp in length.
Further, the sequence of the upstream amplification primer is SEQ ID NO. 3, and preferably, the sequence of the downstream amplification primer is SEQ ID NO. 4.
Further, the RNA is total RNA, mRNA, lncRNA, or ribosome-deleted RNA.
By applying the technical scheme of the invention, RNA is subjected to reverse transcription by adopting a reverse transcription primer with a known sequence and a random sequence, a first sequencing primer sequence is introduced while first-strand cDNA is synthesized, then a second sequencing primer sequence is introduced by directly performing single-link joint connection through cyclase, and finally a proper amplification primer is designed according to the first sequencing primer sequence and the second sequencing primer sequence to perform PCR amplification on a joint connection product, so that an RNA library can be obtained. Compared with the prior art, the method has the advantages that the single-chain joint is connected with the specific first-chain cDNA, so that the synthesis of the second-chain cDNA is not needed, and the chain specificity is directly realized without other digestion processes, so the operation is simple, and a large amount of working hours are saved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a schematic flow diagram of a method for RNA library construction according to the present invention;
FIG. 2A shows the library profile of the library obtained by RNaseH enzyme digestion of yeast RNA samples at the time of library construction (ex-pool concentration 0.532 ng/. Mu.L, volume 10. Mu.L);
FIG. 2B shows a library profile of a yeast RNA sample obtained without RNaseH enzyme digestion at the time of library construction (ex-warehouse concentration 6.2 ng/. Mu.L, ex-warehouse concentration 10. Mu.L);
FIGS. 3A and 3B show the results of the library test of the RNA library of tobacco samples constructed by the conventional method and the method of the present invention in example 2, respectively;
FIG. 4 is a graph showing the difference in gene expression in the RNA library of tobacco samples constructed in example 2 by the conventional method and the method of the present invention;
FIGS. 5A and 5B show the results of library examination of RNA libraries of human samples constructed by the prior art method and the method of the present invention, respectively, in example 3;
FIG. 6 is a graph showing the difference in gene expression in the RNA library of human samples constructed by the conventional method and the method of the present invention in example 3;
FIGS. 7A and 7B show the results of library examination of an Arabidopsis thaliana sample RNA library constructed by the conventional method and the method of the present invention in example 4, respectively;
FIG. 8 is a graph showing the difference in gene expression in the RNA library of Arabidopsis thaliana samples constructed by the conventional method and the method of the present invention in example 4;
FIG. 9 shows the results of the library examination after purification of the yeast sample-TSO primer pool-building-PCR product in example 5 (ex-pool concentration 2.12 ng/. Mu.L, total 15. Mu.L);
FIG. 10 shows the results of the library test after the yeast sample-TSO primer library construction-400-500 bp fragment screening in example 5 (the ex-warehouse concentration is 0.648 ng/. Mu.L, total 10. Mu.L);
FIG. 11 shows the results of the Arabidopsis thaliana sample-TSO primer pooling assay in example 5 (0.9 Xpurification at a stock-removal concentration of 6.0 ng/. Mu.L, total 10. Mu.L, without secondary fragment screening);
FIG. 12 shows the result of human sample-TSO primer pooling test in example 5 (0.906 ng/. Mu.L concentration of pool after fragment screening, 10. Mu.L total, no secondary fragment screening).
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background, the existing RNA library construction method is cumbersome, and in order to improve this situation, in an exemplary embodiment of the present application, a method for constructing an RNA library is provided, as shown in fig. 1, the method comprises: carrying out reverse transcription on the fragmented RNA by adopting a reverse transcription primer to obtain first-strand cDNA; adopting cyclase to carry out single-chain linker connection on the first-chain cDNA to obtain a connection product; and carrying out PCR amplification on the ligation product to obtain an RNA library, wherein the reverse transcription primer sequentially comprises from the 5 'end to the 3' end: known sequences and random sequences.
The method for constructing the RNA library comprises the steps of performing reverse transcription on RNA by using a reverse transcription primer with a known sequence and a random sequence, simultaneously synthesizing first strand cDNA, introducing a first sequencing primer sequence, and then directly performing single-strand ligation by using a cyclase to introduce a second sequencing primer sequence (the method does not comprise an additional step of digesting RNA in an RNA/DNA complex by RNAse H, and the inventor tries to perform single-strand ligation by using and not using RNAse H, and also finds that the library can be successfully constructed without using RNAse H treatment, the concentration of the library is higher, and the library detection result is better, so that the invention does not use RNAse H treatment before linker ligation, and the library detection graph of the prepared library by using and not using RNAse H enzyme to digest cDNA is shown in figures 2A and 2B), and finally performing PCR amplification on the linker ligation product of a proper amplification primer designed according to the first sequencing primer sequence and the second sequencing primer sequence to obtain the RNA library. Compared with the prior art, the method has the advantages that the single-chain joint is connected with the specific first-chain cDNA, so that the synthesis of the second-chain cDNA is not needed, and the chain specificity is directly realized without other digestion processes, so the operation is simple, and a large amount of working hours are saved.
To further improve the efficiency of library construction, in a preferred embodiment, the reverse transcription primer comprises, in order from the 5 'end to the 3' end: spacer arms, known sequences, and random sequences. In some preferred embodiments, the random sequence is a random sequence of 4 to 8 bases, more preferably a random sequence of 6 bases. In other preferred embodimentsIn the examples, the Spacer is Spacer C12, spacer C6, spacer C3, NH 2 And ddC.
The known sequence in the reverse transcription primer can be set by itself, for example, designed according to the sequence of a sequencing primer commonly used by different sequencing platforms. The specific length can also be reasonably set according to the requirement, for example, the length can be set to any length between 16-45bp, for example, 18bp, 20bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp, 40bp, 41bp, 42bp, 43bp, 44bp, 45bp and the like.
The random sequence in the reverse transcription primer is a random sequence consisting of 4 to 8N bases, and N may be any of A, T, C, G. The reverse transcription primer 5' end contains Spacer 6 and other Spacer arm modifications, which can prevent synthesized cDNA from self-linking in the linker ligation reaction, so that the cyclase can only complete the intermolecular ligation of the linker and the cDNA, and any Spacer arm capable of playing the above role is suitable for the application, including but not limited to the above. In a preferred embodiment, the reverse transcription primer has the sequence of SEQ ID NO. 1.
The cyclase is adopted for connection, so that the single-chain linker can be connected with the synthesized first-chain cDNA conveniently by utilizing the capability of promoting intermolecular connection, and the phenomenon of self-connection of the first-chain cDNA is further reduced or avoided. Thus, any cyclase having this function is suitable for use in the present application. In a preferred embodiment, the cyclase is cyclase II or T4 RNA ligase 1. Single-stranded intermolecular ligation preferably employs cyclase II, which is completed in 1 hour. T4 RNA ligase 1 may also be used, but overnight ligation is required for a relatively long time.
In a preferred embodiment, the single-stranded linker has an adenylation modification at the 5 'end and a ddC modification at the 3' end; more preferably, the sequence of the single-stranded linker is SEQ ID NO 2. The 5 'end of the single-stranded linker contains an adenylation modification ensuring ligation to the 3' end of the first-strand cDNA; also, the 3' end of the linker is modified with ddC to prevent self-ligation of the linker.
The amplification primers used in the PCR amplification step can be reasonably designed according to the sequences of the reverse transcription primers and the single-chain linker and by combining with the universal sequence of a sequencing platform and the like. In a preferred embodiment, the ligation product is subjected to PCR amplification by using an upstream amplification primer and a downstream amplification primer to obtain an RNA library, wherein the upstream amplification primer comprises, in order from 5 'end to 3': the sequence of the upstream sequencing platform, the sequence of the first library tag and the sequence of the upstream sequencing primer, wherein the downstream amplification primer sequentially comprises from 5 'end to 3' end: the sequence of the downstream sequencing platform, the sequence of the second library tag and the sequence of the downstream sequencing primer, wherein the sequence of the downstream sequencing primer is the same as at least part of the sequence of the 5 'end of the reverse transcription primer, and the sequence of the upstream sequencing primer is the same as at least part of the sequence of the 5' end of the single-stranded linker; more preferably, the first library tag sequence and the second library tag sequence are each 4 to 10bp in length, most preferably 6 to 8bp in length.
In a preferred embodiment, the sequence of the upstream amplification primer is SEQ ID NO. 3, and more preferably, the sequence of the downstream amplification primer is SEQ ID NO. 4.
In the above construction method, the RNA includes, but is not limited to, total RNA, mRNA, incRNA or ribosome-removing RNA.
The advantageous effects of the present application will be further described with reference to specific examples.
Example 1
1. One-step method for completing RNA fragmentation and reverse transcription reaction (NuoWei Zan kit, cat # NR 604)
(1) Eluting the enriched RNA sample by using 19.5 mu L of fragmentation buffer (Fragment buffer), heating for 8 minutes at 94 ℃ to complete the RNA fragmentation reaction, and immediately carrying out reverse transcription reaction;
(2) The first strand cDNA synthesis reaction system was prepared as follows:
table 1:
(3) First strand cDNA synthesis reactions were performed in a PCR instrument:
table 2:
(4) Immediately purified with 1.8 XDNA Clean magnetic beads (Clean beads) followed by 16. Mu.L ddH 2 And O, eluting the magnetic beads.
2 Single Link Joint connection
(1) The reaction solution was set up as follows:
table 3:
incubate at 60 ℃ for 1h, and incubate at 80 ℃ for 10min for denaturation.
(2) The ligation product was purified using magnetic beads (DNA Clean beads 1.2X), the beads were eluted with 19.15. Mu.L of water, and 18.15. Mu.L were taken for downstream PCR amplification reaction (note: do not aspirate into magnetic beads) (this purification step can remove short-chain reverse transcription primers, linker, etc.).
PCR amplification and purification of the library
(1) Preparing a PCR reaction system:
table 4:
| components | 25 reaction System (μ L) | Final concentration. |
| Template DNA | 18.15 | N/A |
| 5X KAPA HiFi buffer (high fidelity) | 5.0 | 1X |
| 10mM KAPA dNTP Mix | 0.75 | 0.3 mM/piece |
| 25 μ M Forward primer | 0.3 | 0.3μM |
| 25 μ M reverse primer | 0.3 | 0.3μM |
| U/. Mu.L KAPA HiFi Hot Start DNA polymerase | 0.5 | 0.5U |
(2) The PCR reaction was performed according to the following procedure:
table 5:
and (5) detecting the concentration of the sample after the sample is taken off the machine.
(3) Fragment screening was performed using 0.6X and 0.15X DNA Clean beads
Firstly, taking the magnetic beads out of a refrigerator at 4 ℃, uniformly mixing, placing at room temperature for 30min, and complementing the volume of the library to 50 mu L by adopting EB; adding 30 μ L (50 × 0.6 ×) magnetic beads into the sample, mixing, standing at room temperature for 5min, standing on magnetic frame for 5min, collecting supernatant, and removing magnetic beads; taking out 80 μ L of supernatant, adding 7.5 μ L (50X 0.15X) of magnetic beads, mixing, standing at room temperature for 5min, standing on magnetic frame for 5min, keeping magnetic beads, and removing supernatant; adding 200 mu L of freshly prepared 80% ethanol into the magnetic beads, standing for 30s, and removing the supernatant; repeating the step (4) once, and airing the magnetic beads at room temperature for about 3min; adding 15 mu L of EB into the magnetic beads, fully and uniformly mixing, standing for 5min at room temperature, placing for 5min on a magnetic frame, taking 14 mu L of supernatant, and absorbing the concentration of 1 mu L of detection sample.
The primer and linker sequence information used in this example is shown in the following table:
table 6:
and annotating:
(a) The sequence modification method comprises the following steps: spc6, spacer C6 phosphoramidite (Spacer 6-carbon phosphoramidite); rApp, adenylation; ddC, dideoxycytosine (dideoxycytidine);
(b)d，DNA；[N] 6 random hexamer base, N can be any one of A, T, G, C;
(c) The PCR primers contained 8bp index for data resolution.
Wherein the RT primer is a reverse transcription primer, wherein the bold sequence is the same as the bold sequence in PCR primer 1, and correspondingly, the underlined sequence in the linker sequence is the same as the underlined sequence in PCR primer 2.
Example 2
In comparison with the conventional RNA-seq library construction method (i.e., the method of constructing a library by synthesizing double-stranded cDNA mentioned in the background), the quality control results of the libraries constructed by the two methods are shown in FIG. 3A and FIG. 3B, respectively, when the method of the present invention is used for testing in tobacco samples.
The sequencing data analysis results are shown in Table 7, Q20 and Q30 are respectively more than 97% and more than 92%, the mapping rate is more than 94%, and other indexes meet the requirements. FIG. 4 shows a graph of the differences in gene expression detected after library construction by the two methods, from which it can be seen that the method of the present invention is consistent with the results of the conventional method.
Table 7:
example 3:
compared with the common RNA-seq library construction method, the quality inspection results of the libraries constructed by the two methods are respectively shown in FIG. 5A and FIG. 5B when the method of the invention is used for testing in human samples.
The sequencing data analysis results are shown in Table 8, Q20 and Q30 are respectively more than 96% and more than 91%, the mapping rate is more than 95%, and other indexes all meet the requirements. FIG. 6 shows a graph of the differences in gene expression detected after library construction by the two methods, from which it can be seen that the method of the present invention is consistent with the results of the conventional method.
Table 8:
example 4:
compared with the common RNA-seq library construction method, the quality inspection results of the libraries constructed by the two methods are respectively shown in FIG. 7A and FIG. 7B when the method is used for testing in an Arabidopsis sample.
The sequencing data analysis results are shown in table 9, and show that Q20 and Q30 are respectively more than 96% and more than 91%, the mapping rate is more than 94%, and other indexes meet the requirements. FIG. 8 is a graph showing the difference in gene expression detected after constructing a library by the two methods, and it can be seen that the method of the present invention is consistent with the results of the conventional method.
Table 9:
example 5:
compared with the RNA-seq library construction method disclosed in the embodiment 1 of CN108251503B, the method uses the template conversion principle of Smarter/Smart-seq2 to construct a library, is only suitable for single-cell, micro-scale or ultra-micro library construction (10 pg-10 ng), and is not suitable for high-initial-quantity library construction. We tried Smart-seq2 pooling using total RNA between 100ng and 1000ng, as shown in FIGS. 9 and 10, which are both pooling plots using TSO primers by template switching method, and showed more small fragments of 100-200bp, which were essentially unsuccessful.
To improve the Template conversion efficiency of TSO (Template-Switching oligonucleotide) primers, we added Betaine (5M) to the reverse transcription system and performed experiments with new samples, but the library examination results still showed high library content of small fragments, as shown in FIG. 11 and FIG. 12.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: introducing a first sequencing primer sequence in the reverse transcription process through a reverse transcription primer, simultaneously using the intermolecular connection capacity of the cyclase II to connect a joint with a cDNA chain, introducing a second sequencing primer sequence, designing a PCR primer according to the two sequencing primer sequences to amplify the library, and finally completing the library construction. It should be noted that the method of the present invention is particularly suitable for the construction of eukaryotic RNA libraries.
Compared with the existing transcriptome sequencing and database building method, the method has the following advantages:
(1) The invention uses a one-step method to complete RNA fragmentation and cDNA strand synthesis, compared with the prior art, the time is saved, and the specificity of strands is distinguished without using uracil U base to carry out cDNA double strand synthesis; the cDNA synthesized by reverse transcription of the invention is derived from mRNA, namely, the specificity of the chain is highlighted;
(2) The invention directly connects the cDNA after reverse transcription by a joint, the 5 'end of the joint contains adenylation modification, and the connection with the 3' end of the cDNA is ensured; meanwhile, the 3' end of the joint contains ddC modification to prevent self-connection; the 5' end of the RT primer contains Spc6 modification to prevent the synthesized cDNA from self-ligation in a linker ligation reaction, so that the cyclase can only complete intermolecular ligation of the linker and the cDNA; compared with the prior art, the process of repairing the tail end and adding the adenine A base for connection is omitted, the operation flow is reduced, and the time is saved.
(3) Compared with the prior art, the method uses a single-strand connection mode to establish the library, does not need the process of annealing and synthesizing double-strand connectors, and does not need the process of adding USER enzyme to digest cDNA double-strand containing uracil U basic groups before PCR amplification, so that the operation is simpler and more efficient.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (10)
1. A method of constructing an RNA library, comprising:
performing reverse transcription on the fragmented RNA by using a reverse transcription primer to obtain first strand cDNA, wherein the reverse transcription primer sequentially comprises from 5 'end to 3' end: known sequences and random sequences;
performing single-chain linker connection on the first-chain cDNA by using cyclase to obtain a connection product;
performing PCR amplification on the ligation product to obtain the RNA library;
the cyclase is cyclase II or T4 RNA ligase 1, the 5 'end of the single chain link is provided with adenylation modification, the 3' end of the single chain link is provided with ddC modification, and the sequence of the single chain link is SEQ ID NO. 2.
2. The method of claim 1, wherein the reverse transcription primer further comprises a spacer arm upstream of the known sequence.
3. The method of claim 2, wherein the random sequence is 4~8 bases.
4. The method of claim 2, wherein the random sequence is a 6-base random sequence.
5. The method according to claim 2, wherein the Spacer is Spacer C12, spacer C6, spacer C3, NH 2 And ddC.
6. The method according to any one of claims 1 to 5, wherein the reverse transcription primer has the sequence of SEQ ID NO. 1.
7. The method for constructing the RNA library according to claim 1, wherein the ligation product is subjected to PCR amplification by using an upstream amplification primer and a downstream amplification primer to obtain the RNA library, and the upstream amplification primer sequentially comprises from 5 'end to 3' end: the sequence of the upstream sequencing platform, the sequence of the first library tag and the sequence of the upstream sequencing primer, wherein the downstream amplification primer sequentially comprises from 5 'end to 3' end: a downstream sequencing platform sequence, a second library tag sequence, and a downstream sequencing primer sequence, wherein the downstream sequencing primer sequence is identical to at least a portion of the sequence at the 5 'end of the reverse transcription primer, and the upstream sequencing primer sequence is identical to at least a portion of the sequence at the 5' end of the single-stranded linker.
8. The construction method according to claim 1,
the lengths of the first library tag sequence and the second library tag sequence are respectively 4-10bp.
9. The construction method according to claim 8, wherein the sequence of the upstream amplification primer is SEQ ID NO. 3,
the sequence of the downstream amplification primer is SEQ ID NO. 4.
10. The method according to claim 1, wherein the RNA is total RNA, mRNA, incRNA, or ribosome-deleted RNA.
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| US8617817B2 (en) * | 2010-02-12 | 2013-12-31 | Genisphere, Llc | Whole transciptome sequencing |
| US10316356B1 (en) * | 2014-11-21 | 2019-06-11 | Mgi Tech Co., Ltd. | Method of constructing sequencing library with bubble-shaped adaptor element |
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