阅读说明：本技术 Rna建库方法 (RNA library construction method ) 是由 李新 马玉 李瑞强 赵桂仿 于 2020-12-03 设计创作，主要内容包括：本申请提供了一种RNA建库方法。该建库方法包括：利用poly(dT)与ploy(A)的结合捕获富集mRNA；对mRNA进行片段化处理,得到片段化mRNA；对片段化mRNA进行逆转录,得到片段化双链cDNA；对片段化双链cDNA进行文库构建,得到降解RNA测序文库。通过利用poly(dT)与ploy(A)结合的方式富集mRNA后,先对富集的mRNA进行片段化处理,然后逆转录合成双链cDNA,之后利用经典的末端修复加A和TA碱基连接方式建库,提高对降解RNA的兼容性,使得该建库方法能够适合任意样本的RNA文库构建。该建库方法对降解RNA文库构建及产出数据质量的提高效果更显著。(The present application provides a method for RNA library construction. The database building method comprises the following steps: capturing the enriched mRNA using poly (dT) in combination with ploy (A); fragmenting mRNA to obtain fragmented mRNA; reverse transcription is carried out on the fragmented mRNA to obtain fragmented double-chain cDNA; and constructing a library of the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library. After mRNA is enriched by combining poly (dT) and ploy (A), the enriched mRNA is firstly fragmented, then is subjected to reverse transcription to synthesize double-stranded cDNA, and then is subjected to library construction by using a classical terminal repair and A and TA base connection mode, so that the compatibility of degraded RNA is improved, and the library construction method can be suitable for RNA library construction of any sample. The database building method has more remarkable effect on the construction of the degraded RNA library and the improvement of the quality of output data.)

1. An RNA library construction method, comprising:

capturing the enriched mRNA using poly (dT) in combination with ploy (A);

fragmenting the mRNA to obtain fragmented mRNA;

reverse transcription is carried out on the fragmented mRNA to obtain fragmented double-chain cDNA;

and constructing a library of the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library.

2. The library construction method of claim 1, wherein capturing enriched mRNA using poly (dt) in combination with ploy (a) comprises:

capturing and enriching mRNA in the total RNA by using poly (dT) -containing magnetic beads to obtain a magnetic bead-mRNA compound;

preferably, the total RNA is total RNA in the presence of degradation, more preferably total RNA with a RIN value of 7 or less.

3. The library construction method of claim 2, wherein capturing enriched mRNA using poly (dt) in combination with ploy (a) comprises:

denaturing the total RNA at a first temperature to open secondary structures;

mixing the magnetic beads with poly (dT) with the total RNA after the secondary structure is opened to obtain a mixture;

subjecting the mixture to a second temperature to allow the poly (dT) to bind to ploy (A) for a first capture of mRNA;

placing the magnetic beads after the first capture at a third temperature to release the first captured products;

after the magnetic beads with the released first captured products are processed, the poly (dT) is combined with ploy (A) again to capture mRNA for the second time;

preferably, the first temperature is 60-68 ℃, and more preferably 65 ℃;

preferably, the second temperature is 20-27 ℃, and more preferably 25 ℃;

preferably, the third temperature is 78-85 ℃, and more preferably 80 ℃.

4. The library construction method of claim 2 or 3, wherein the mRNA is fragmented, and obtaining fragmented mRNA comprises:

incubating and interrupting the magnetic bead-mRNA compound and a fragmentation buffer solution at 90-95 ℃ to obtain the fragmented mRNA;

preferably, the time for interrupting the incubation is 10-20 min.

5. The library construction method of claim 4, wherein performing library construction on the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library comprises:

performing end repair and A addition on the fragmented double-stranded cDNA to obtain repair DNA;

connecting the repair DNA with a linker with a molecular marker to obtain a connection product;

and carrying out PCR amplification on the connection product to obtain the degraded RNA sequencing library.

6. The library-building method of claim 5, wherein the linker with the molecular marker is a Y-linker with the molecular marker at both ends.

7. The library construction method of claim 6, wherein the Y-linker comprises linker 1 and linker 2, and the sequence of linker 1 is as shown in SEQ ID NO: 1, the sequence of the joint 2 is shown as SEQ ID NO: 2, respectively.

8. The library construction method according to claim 6, wherein the molecular tag in the linker with molecular tag is a random nucleic acid sequence of 4-8 nt, and the molecular tag of at least one end of any 2Y-linkers is different;

preferably, the molecular marker is any one of table 2.

9. The library construction method according to claim 5, wherein the library construction method further comprises a step of fragment screening of the ligation products before PCR amplification of the ligation products; preferably, magnetic beads are used for fragment screening.

10. The library construction method of claim 5, wherein reverse transcribing the fragmented mRNA to obtain fragmented double-stranded cDNA comprises:

performing reverse transcription on the fragmented mRNA to synthesize first strand cDNA;

synthesizing second strand cDNA by dNTP containing dUTP to obtain fragmented double strand cDNA;

preferably, the ligation product is digested with USER enzyme to remove the U base-containing second strand cDNA prior to PCR amplification of the ligation product.

Technical Field

The application relates to the field of sequencing library construction, in particular to an RNA library construction method.

Background

RNA-seq is an important tool for researching gene expression of a eukaryotic transcriptome, and in recent years, an absolute quantitative RNA-seq library construction method and a kit can correct the preference caused by PCR amplification by adding a molecular tag (UMI) to a reverse-transcribed cDNA Molecule, so as to achieve the effect of accurately quantifying the gene expression of the transcriptome; meanwhile, the wrong base generated in the sequencing process can be corrected. However, the existing method and kit have high requirements on RNA quality, and degraded RNA samples are difficult to ensure the quality of sequencing data.

For example, the kit for the Wuhankang detection technology requires that the RIN value of RNA is 7, the agarose electrophoresis 28S:18S ratio is more than 1.5, and the kit has no dragging and the like. The principle is that a Reverse Transcription primer (RT) is introduced into a PCR upstream site in the Reverse Transcription process; subsequently, introducing UMI and PCR downstream sites in the first strand of cDNA by means of single-stranded intermolecular ligation; finally, library construction was completed by PCR amplification and purification.

It can be seen that the existing kit is not suitable for RNA samples with low degradation or RIN value, and the generated sequencing results have the problems of high repeated data occupation ratio (high Dup rate), low effective data occupation ratio (low Clean Reads), large amount of linker pollution and the like, especially in the large-scale nucleic acid extraction process, under the condition that it is difficult to ensure that high-purity and high-RIN value RNA is obtained, the method or the kit is not suitable for the requirement of industrialized sequencing library building.

Content of application

The application mainly aims to provide an RNA library construction method to solve the problem that the existing method in industrialized sequencing library construction is not suitable for degrading RNA sample library construction and sequencing in the prior art.

In order to achieve the above object, according to one aspect of the present application, there is provided an RNA database construction method comprising: capturing the enriched mRNA using poly (dT) in combination with ploy (A); fragmenting mRNA to obtain fragmented mRNA; reverse transcription is carried out on the fragmented mRNA to obtain fragmented double-chain cDNA; and performing library construction on the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library.

Further, capturing and enriching mRNA in the total RNA by using magnetic beads with poly (dT) to obtain magnetic bead-mRNA complexes; preferably, the total RNA is total RNA in the presence of degradation, more preferably total RNA with a RIN value of 7 or less.

Further, capturing enriched mRNA using poly (dt) in combination with ploy (a) comprises: denaturing the total RNA sample at a first temperature (preferably 60-68 ℃, more preferably 65 ℃) to open secondary structures; then, mixing the magnetic beads with poly (dT) with the total RNA after opening the secondary structure to obtain a mixture; allowing poly (dT) to bind to ploy (A) at a second temperature (preferably 20-27 ℃, more preferably 25 ℃) to capture the mRNA for the first time; releasing the first captured product from the magnetic beads at a third temperature (preferably 78-85 ℃, more preferably 80 ℃); after the magnetic beads after releasing the first captured product are processed (i.e., binding buffer is added), poly (dt) is again bound to ploy (a) to capture mRNA a second time.

Further, fragmenting the mRNA to obtain fragmented mRNA includes: incubating and interrupting the magnetic bead-mRNA compound and the fragmentation buffer solution at 90-95 ℃ to obtain fragmented mRNA; preferably, the time for interrupting the incubation is 10-20 min.

Further, performing library construction on the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library, wherein the library comprises: carrying out end repair and A addition on the fragmented double-stranded cDNA to obtain repair DNA; connecting the repair DNA with a linker with a molecular marker to obtain a connection product; and carrying out PCR amplification on the connection product to obtain a degraded RNA sequencing library.

Further, the linker with the molecular marker is a Y-type linker with the molecular marker at both ends.

Further, the Y-type linker comprises a linker 1 and a linker 2, and the sequence of the linker 1 is shown in SEQ ID NO: 1, the sequence of the linker 2 is shown as SEQ ID NO: 2, respectively.

Further, the molecular markers in the joints with the molecular markers are random nucleic acid sequences of 4-8 nt, and the molecular markers at least one end of any 2Y-shaped joints are different; preferably, the molecular marker is any one of table 2.

Further, before PCR amplification of the ligation product, the library construction method further comprises a step of fragment screening of the ligation product; preferably, magnetic beads are used for fragment screening.

Further, reverse transcription of the fragmented mRNA to obtain fragmented double-stranded cDNA comprises: carrying out reverse transcription on the fragmented mRNA to synthesize first strand cDNA; synthesizing second strand cDNA by dNTP containing dUTP to obtain fragmented double strand cDNA; preferably, the ligation product is digested with USER enzyme to remove the U base-containing second strand cDNA prior to PCR amplification of the ligation product.

According to the technical scheme, after mRNA is enriched by combining poly (dT) and ploy (A), the enriched mRNA is fragmented, then double-stranded cDNA is synthesized by reverse transcription, and then a library is built by using a classical terminal repair and A and TA base connection mode, so that the compatibility of degraded RNA is improved, and the library building method can be suitable for the construction of an RNA library of any sample. The database building method has more remarkable effect on the construction of the degraded RNA library and the improvement of the quality of output data.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate exemplary embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:

FIG. 1 shows a schematic flow diagram of the degraded RNA library construction method of the present application;

FIG. 2 shows statistics of the number of identical reads molecules in sequencing data for different species in the examples of the present application;

FIG. 3 shows a gel electrophoresis of human sample degradation in an embodiment of the present application;

FIG. 4 shows the results of H7667 sample library examination in the examples of the present application;

FIG. 5 shows the result of H7672 sample library examination in the example of the present application;

FIG. 6 shows the result of H7740 sample library examination in the embodiment of the present application;

fig. 7 shows the results of H7617 sample library examination in the example of the present application.

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 application will be described in detail with reference to examples.

Aiming at the existing conditions in the prior art, the invention researches an absolute quantitative library construction method suitable for RNA sample sequencing of degraded or low RIN value (such as degraded samples with RIN value below 7), and the data results of the library construction method have no difference with normal or high RIN value samples, so that the method is suitable for sequencing and library construction of RNA samples with different qualities obtained by large-scale nucleic acid extraction, and meets the requirement of industrialized sequencing and library construction.

On the basis of the results of this study, the applicant proposed the solution of the present application. In a preferred embodiment, there is provided an RNA banking method comprising: capturing the enriched mRNA using poly (dT) in combination with ploy (A); fragmenting mRNA to obtain fragmented mRNA; reverse transcription is carried out on the fragmented mRNA to obtain fragmented double-chain cDNA; and constructing a library of the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library.

According to the RNA library construction method provided by the application, after mRNA is enriched by using a mode of combining poly (dT) and ploy (A), the enriched mRNA is firstly subjected to fragmentation treatment, then reverse transcription is carried out to synthesize double-stranded cDNA, and then a library is constructed by using a classical terminal repair and A and TA base connection mode, so that the compatibility of degraded RNA is improved, and the library construction method can be suitable for constructing RNA libraries of any samples. The database building method has more remarkable effect on the construction of the degraded RNA library and the improvement of the quality of output data.

In a preferred embodiment, poly (dT) -containing magnetic beads are used to capture and enrich mRNA in total RNA, and magnetic bead-mRNA complexes are obtained. A specific kind of magnetic beads may be an existing product such as kit of ABConal (RK 20302).

In a preferred embodiment, capturing enriched mRNA using poly (dt) in combination with ploy (a) comprises: denaturing the total RNA at a first temperature, and then mixing magnetic beads with poly (dT) with the total RNA to obtain a mixture; allowing poly (dT) to bind to ploy (A) to perform a first capture of the mRNA, resulting in a first capture product; releasing the first capture product from the magnetic beads at a second temperature, and then treating the magnetic beads to allow poly (dT) to bind to ploy (A) again to capture the mRNA a second time; preferably, the first temperature is 60-68 ℃, and optimally 65 ℃; preferably, the second temperature is 20-27 ℃, and most preferably 25 ℃; preferably, the third temperature is 78-95 ℃, and most preferably 80 ℃.

In a preferred embodiment, fragmenting the mRNA to obtain fragmented mRNA comprises: incubating and interrupting the magnetic bead-mRNA compound and the fragmentation buffer solution at 90-95 ℃ to obtain fragmented mRNA; more preferably, the time for interrupting the incubation is 10-20 min.

The mRNA is separated by incubation and breaking at 90-95 ℃ to break, and fragmented mRNA is formed, so that the size of a target fragment required by on-machine sequencing is obtained. Here, the mRNA after fragmentation refers to an mRNA disruption sequence in the supernatant that is not adsorbed to the magnetic beads. Subsequent reverse transcription synthesis was performed by taking fragmented mRNA from the supernatant.

In a preferred embodiment, library construction of the fragmented double-stranded cDNA to obtain a degraded RNA sequencing library comprises: performing end repair and A addition on the fragmented double-stranded cDNA to obtain repair DNA; connecting the repair DNA with the linker labeled with the band molecules to obtain a connection product; and carrying out PCR amplification on the connection product to obtain a degraded RNA sequencing library.

The RNA library construction method has the advantages that the RNA library construction universality and success rate of samples from different sources can be improved by adopting a classical TA connection mode to carry out library construction.

In a preferred embodiment, the linker with the molecular marker is a Y-type linker with the molecular marker at both ends.

In a preferred embodiment, the Y-linker comprises linker 1 and linker 2, and the sequence of linker 1 is as set forth in SEQ ID NO: 1, the sequence of the linker 2 is shown as SEQ ID NO: 2, respectively.

In a preferred embodiment, the molecular marker in the linker with molecular marker is a random nucleic acid sequence of 4-8 nt, and the molecular marker at least one end of any 2Y-type linkers is different; more preferably, the molecular marker is any one of table 2.

In the above preferred embodiment, by introducing a molecular marker (UMI) on the cDNA molecule by TA base ligation, the molecular marker will accompany the whole process of PCR amplification and on-machine sequencing, the generated sequencing data can be used to quantify gene expression abundance by calculating UMI and correct the erroneous base randomly generated during sequencing, thus being an absolute quantitative library construction method for RNA samples, especially for degraded or low-quality RNA.

It should be noted that the UMIs used in the current methods involving absolutely quantitative transcriptome are all random bases, for example, UMIs composed of 10 to 12N (any of A, T, G, C), but in the double-strand annealing process, it is difficult to synthesize complementary double strands that match each other efficiently, many are mismatched sequences, the yield of the method by gel cutting recovery is very low, and the method cannot meet the industrial requirements at all, and the cost is high. The molecular marker in the application is directly synthesized UMI with a fixed known sequence, then is annealed into double chains, and then the double chains UMI are mixed for use, so that the use amount is ten thousand times enough, the stability is good, and the RNA degradation is compatible. In addition, the application also compares a health test kit adopting random UMI, and the result shows that the performance of the kit is unstable and the GC curve fluctuates greatly. Moreover, regarding the UMI joint, the method for preparing the double-chain UMI joint by using enzyme digestion is also carried out in the application, and the library building result is consistent with the result of the invention, but the method for preparing the UMI joint is too complicated, the UMI joint is prepared for only 5-20 times, and the UMI joint is not modified and has degradation risk.

The Y-type joint containing 80 modified different molecular markers shown in the table 2 is adopted, 6400 UMIs are connected at two ends of the Y-type joint, the number of the same cDNA molecules can be completely covered, and the modification of a joint sequence can also ensure the stability of T base suspension.

In a preferred embodiment, the library construction method further comprises the step of fragment screening the ligation products prior to PCR amplification of the ligation products; preferably, magnetic beads are used for fragment screening.

In a preferred embodiment, the U-containing second strand cDNA in the ligation product is digested and degraded using the USER enzyme. dUTP is used for synthesizing the cDNA double strand, and the cDNA of the second strand can be removed by USER enzyme treatment before PCR, thereby achieving the purposes of strand-specific amplification and strand-specific library construction.

The advantageous effects of the present application will be further described with reference to specific examples. In the following examples, the mRNA capture kit was ABConal's kit (RK20302), and the other library construction-related reagents were self-prepared reagents (Novo reagents) produced from Nordheim.

The following examples were carried out according to the flow shown in fig. 1, and the main steps are as follows, and are specifically described in example 1.

1) mRNA capture of Poly A tail was accomplished using the kit of ABConal (RK20302), mRNA containing Poly A tail was captured by Oligo d (T) magnetic beads, supernatant was discarded after repeated washing, nucleic acid was retained on the beads and disrupted by incubation with fragmentation buffer at 94 ℃ for 15 min, followed immediately by reverse transcription to double-stranded cDNA;

2) using a self-prepared terminal repair system and a PCR amplification system to carry out cDNA terminal repair, A base addition and PCR amplification, and connecting by using a UMI joint mixed in advance;

3) the number of PCR amplification cycles is 13-16; after the PCR reaction is finished, the fragment with the range of 300-500bp is immediately purified by using the whole gold magnetic bead.

Example 1

1.1 mRNA Capture

1.1.1 reagent preparation

Poly (A) mRNA Purification Module was removed from the reaction mixture at 4 ℃ and allowed to stand to equilibrate to room temperature.

1.1.2 RNA sample preparation

In a nucleic-free PCR tube, 0.1-4. mu.g of RNA sample was diluted to 50. mu.l with nucleic-free water and placed on ice for use.

1.1.3 magnetic bead preparation

(1) Vortex and mix Oligo d (T)25 captured magnetic beads, take 20 μ L into 1.5ml EP tube, add 200 μ L mRNA binding buffer solution, blow and mix;

(2) placing 1.5ml EP tube on magnetic frame for about 2min until the solution becomes clear, and discarding the supernatant; adding 200 μ L of mRNA binding buffer solution, blowing, stirring, standing for 2min until the solution becomes clear, and removing the supernatant;

(3) taking down a 1.5ml EP tube, adding 50 mul mRNA binding buffer solution, blowing, beating and mixing evenly; add 50. mu.l of magnetic beads to the total RNA sample and pipette 6 times to mix thoroughly.

1.1.4 RNA denaturation and Capture

(1) Mu.l of the sample was placed in a PCR instrument and incubated at 65 ℃ for 5min to denature the RNA.

(2) The mixture was left at room temperature of 25 ℃ for 5min to bind mRNA to the magnetic beads.

(3) Placing the sample in a magnetic frame for 2min to separate mRNA from total RNA; the supernatant was carefully removed.

(4) The sample was removed from the magnetic stand, flushed 6 times with 200. mu.l of Washing buffer to thoroughly mix, allowed to stand on the magnetic stand for 5min, and the supernatant carefully removed.

(5) Taking the sample out of the magnetic frame, and taking 50 mu l of Tris buffer solution to resuspend the magnetic beads; the mixture was thoroughly mixed by pipetting 6 times.

(6) Placing the sample in a PCR instrument, keeping the temperature at 80 ℃ for 2min, standing at room temperature for 2min, and eluting mRNA.

(7) Add 50. mu.l of mRNA binding buffer and pipette 6 times to mix thoroughly.

(8) The mixture was allowed to stand at room temperature for 5min to bind mRNA to the magnetic beads.

(9) Placing the sample in a magnetic frame for 5min to separate mRNA from total RNA; the supernatant was carefully removed.

(10) The sample was removed from the magnetic stand, flushed 6 times with 200 μ l Washing buffer to thoroughly mix, left on the magnetic stand for 5min, carefully removed all the supernatant, and removed again after transient centrifugation.

(11) The 1 XFrag/Elute buffer was prepared as follows:

table 1:

(12) add 10.4. mu.l of Frag/Elute buffer, pipette well, and incubate at 94 ℃ for 15 minutes to break the RNA (hot lid temperature 105 ℃).

(13) When the temperature is reduced to 4 ℃, the centrifugal tube is taken out, is subjected to instantaneous centrifugation, is placed on a magnetic frame, and after the solution is clarified, 10 mu l of supernatant is taken out to another PCR tube, and then the next step of First strand cDNA synthesis is immediately carried out.

1.1.5 first Strand cDNA Synthesis

(1) The RT Reagent was taken out and dissolved at room temperature, and the following system was prepared on ice:

table 2:

(2) and (3) blowing and beating the mixture uniformly by using a pipettor, performing instantaneous centrifugation, and placing the system into a PCR instrument:

table 3:

1.1.6 second Strand cDNA Synthesis

(1) The second strand synthesis reaction buffer was removed from the refrigerator and the following reaction system was prepared on ice:

table 4:

it should be noted that, in the cDNA double-strand synthesis, dNTP is used as a filling substrate, the complementary strand synthesis is completed under the action of DNA polymerase, dUTP is added here, the synthesized double strand contains U bases, and the subsequent USER enzyme can digest the double strand against the U bases to realize strand-specific amplification and library construction.

(2) Using a pipettor to blow and mix the mixture evenly, placing the sample in a PCR instrument (the temperature of a hot cover is closed) after instantaneous centrifugation,

preserving heat at 16 ℃ for 1h, and performing hold at 4 ℃;

(3) 144. mu.l (1.8X) of whole gold DNA clean beads (equilibrated to room temperature) was put into a 1.5ml EP tube, and 80. mu.l of the above reaction system was added thereto and mixed well. Standing at room temperature for 5min, standing on a magnetic frame for 5min, and discarding the supernatant.

(4) The EP tube was held on the magnetic stand, 200. mu.l of freshly prepared 80% ethanol was added to the EP tube, allowed to stand for 30s, and the supernatant was discarded.

(5) Repeating the step (4), washing the magnetic beads with 80% ethanol for 1 time again, and completely sucking the residual liquid with a 10-microliter gun head; and drying the magnetic beads for 3-5min at room temperature.

(6) The EP tube was removed from the magnetic stand, 43. mu.l of nuclease-free water was added, mixed well, left at room temperature for 5min, left on the magnetic stand for 5min, 42. mu.l of the supernatant was taken in a 0.2ml EP tube and subjected to the next step.

Note: the double-stranded cDNA product may be stored temporarily at-20 ℃ overnight.

1.2 end repair plus A and UMI linker connection:

1.2.1 end repair plus A

(1) The self-prepared repair buffer and repair enzymes (Klenow enzyme + T4 DNA polymerase + T4 PNK) were removed from the refrigerator and the reagents were added sequentially according to the following system:

table 5:

(2) using a pipettor to mix gently and evenly, performing instantaneous centrifugation, and placing the system in a PCR instrument:

table 6:

(3) immediately after the reaction, the reaction mixture was placed on ice for the next linker ligation reaction.

1.2.2 Joint connection

Preparing a Y-shaped joint:

prior to the preparation of Y-linkers, the number of molecules of the same reads in 1ug starting amount of total RNA library of different species was assessed. According to the statistics of the student information analysis, the reads with the number of the top 5 in the sample library of the human, the rabbit, the goat, the rice, the tobacco and the like do not exceed 5000 at most (as shown in figure 2). Therefore, if the number of UMI molecules exceeds the predetermined number, the number of reads can be completely covered, and the purpose of quantification is achieved.

Ordering the following linker at a primer synthesis company, purifying by HPLC to obtain freeze-dried powder, adding water to dissolve the freeze-dried powder, and annealing to synthesize a double-chain linker; wherein nnnnnnn are actually 80 different fixed sequences, and are synthesized in the adaptor 1 respectively, as shown in table 8, and NNNNN in the adaptor 2 is a sequence complementary to NNNNN in the adaptor 1, and the NNNNN sequences of the adaptor 1 and the adaptor 2 are ensured to be complementary sequences, and then annealing is performed; subsequently, the following 80 double-stranded complementary linkers containing different NNNNN bases were mixed equimolar to prepare a linker mixture, the library was double-ended by 80, i.e. 6400 linkers could completely cover the number of molecules of RNA.

Table 7:

joint name	Sequence of
		Joint 1	5'-acactctttccctacacgacgctcttccgatctnnnnn*-t-3′(SEQ ID NO：1)
Joint 2	5’-p-nnnnnagatcggaagagcacacgtctgaactccagtc-3’(SEQ ID NO：2)

*: modification of phosphorothioate linkages

P: phosphorylation modifications

NNNNN is the following sequence information

Table 8:

TACGT

TGAGT

TAACC

CTCAA

CGACG

ACCCC

TCATC

TCGTC

GATTA

GGATC

GCGAG

ATGTG

CCCAA

AGGAA

CGTGT

ACGGA

TTCTA

GTCAT

AGTGA

CTTAC

TTCCT

CAGCC

AATGG

ATTTC

GTCAA

GTCGG

CGCAA

ACTCA

TCAGT

AATCG

AACAG

CGTGG

TGATC

TACCA

GGTTT

CCCGA

CACTC

CACTT

GGGCG

GAATT

GTAAG

CCTAC

GATAC

ACGAA

CCCTG

CCAGT

GACGA

TGCAT

GGCGA

AGGTG

TGGAC

TCTAT

CAGGG

CGTAT

TTGCT

TTATG

GGATG

GTTTT

TCTGA

TTAGA

AAGCT

GCTCA

CTTGC

GACCC

CAGTC

ACAAG

CATCA

TTAGC

GAGCG

TCACC

ACGTT

AGACC

CTATT

AGAGA

TGGCT

GTTCT

ATGAG

GAGGC

ATACG

ACGAG

(1) the attachment system was added as follows:

table 9:

(2) blowing and mixing uniformly by using a pipettor, instantly centrifuging, placing the sample in a PCR instrument (the temperature of a hot cover is closed), and preserving the temperature for 30min at 20 ℃ and hold at 4 ℃;

1.2.3 ligation fragment screening

(1) Add 20.3. mu.l nuclease-free water to the ligation product and make up to 100. mu.l system.

(2) Mu.l (0.3X) of whole gold DNA clean beads (equilibrated to room temperature) was taken in a 1.5ml EP tube, and 100. mu.l of the above reaction system was added thereto and mixed well. Standing at room temperature for 5min, and standing on magnetic frame for 5min until the solution becomes clear (cutting and keeping the supernatant).

(3) The supernatant was transferred to another 1.5ml EP tube, 30. mu.l (0.3X) of whole gold DNA clean beads (equilibrated to room temperature) were added, and vortexed; standing at room temperature for 5min, standing on magnetic frame for 5min, and discarding supernatant.

(4) The EP tube was held on the magnetic stand, 200. mu.l of freshly prepared 80% ethanol was added to the EP tube, allowed to stand for 30s, and the supernatant was discarded.

(5) Repeating the step (4), washing the magnetic beads with 80% ethanol for 1 time again, and completely sucking the residual liquid with a 10-microliter gun head; and drying the magnetic beads for 3-5min at room temperature.

(6) The EP tube was removed from the magnetic frame, 23. mu.l of nuclease-free water was added, mixed well, left for 5min at room temperature, left for 5min on the magnetic frame, 22. mu.l of supernatant was taken in a 0.2ml EP tube and the next step was performed.

1.3 library amplification and fragment screening

(1) And taking the PCR mix, the F primer, the R primer and the USER enzyme out of a refrigerator at the temperature of-20 ℃, and preparing a PCR system on ice.

Table 10:

(2) and (4) blowing, beating, mixing uniformly, performing instantaneous centrifugation, and placing in a PCR instrument.

Table 11:

note: when the sample quality meets the requirement, the amplification can be carried out on 100ng samples and more samples by using 13 cycles. And when the mass concentration and the RIN value of the sample do not meet the requirements, increasing 2-15 cycles.

(3) Add 50.3. mu.l nuclease-free water to the PCR product and make up to 100. mu.l system.

(4) 60. mu.l (0.6X) of whole gold DNA clean beads (equilibrated to room temperature) was put into a 1.5ml EP tube, and 100. mu.l of the above reaction system was added thereto and mixed well. Standing at room temperature for 5min, and standing on magnetic frame for 5min until the solution becomes clear (cutting and keeping supernatant).

(5) The supernatant was transferred to another 1.5ml EP tube, 15. mu.l (0.15X) of whole gold DNA clean beads (equilibrated to room temperature) were added, and vortexed; standing at room temperature for 5min, standing on magnetic frame for 5min, and discarding supernatant.

(6) The EP tube was held on the magnetic stand, 200. mu.l of freshly prepared 80% ethanol was added to the EP tube, allowed to stand for 30s, and the supernatant was discarded.

(7) Repeating the step (4), washing the magnetic beads with 80% ethanol for 1 time again, and completely sucking the residual liquid with a 10-microliter gun head; and drying the magnetic beads for 3-5min at room temperature.

(8) The EP tube was removed from the magnetic stand, 12. mu.l of nuclease-free water was added, mixed well, left at room temperature for 5min, left on the magnetic stand for 5min, and 11. mu.l of the supernatant was taken in a 0.5ml EP library stock tube.

(9) The Qubit value of 1. mu.l of the stock solution of the test library was diluted to 1 ng/. mu.l (at least 10. mu.l) for library detection.

Example 2

The extracted human RNA samples (as shown in fig. 3) with different RIN values or degradation degrees were sequenced and pooled using the method of the present invention and the health test kit, respectively, and the results are shown in table 12: the method of the invention shows good compatibility to RNA samples, and RNAs with different RIN values or degradation degrees show nearly the same data quality (as shown in Table 12) and library peak detection graphs (as shown in FIGS. 4 to 7); in contrast, the assay kit had poor compatibility with low RIN values or degraded RNA, with higher Dup rates and very high linker content in the degraded RNA data (as shown in table 12).

The RNA degradation of the human sample is shown in FIG. 3, and the degradation degree of the other three samples except the H7617 sample is different, wherein the H7667 sample is most severely degraded, and 18S and 28S bands are hardly observed.

Table 12:

example 3:

the goat and haloxylon ammodendron samples are sequenced and subjected to library building by using the method, the initial amount of RNA is 50ng and 100ng respectively, the data analysis result is shown in table 13, and the data result under the initial amount has good output and quality.

Table 13:

example 4:

taking 100ng of goat testis sample RNA as an example, respectively using the linker in the method and the linker prepared by the enzyme cutting method (specific sequences are shown in Table 14) to build a library, wherein the usage amount of the linker is 10ng, the data results are shown in Table 17, the data results have no obvious difference, but the preparation process of the linker by the enzyme cutting method is complicated, the UMI linker needs to be obtained through annealing, extension, enzyme cutting, gel cutting recovery and other processes, and the prepared linker content cannot meet the requirement of large-scale industrialized library building.

Table 14:

the preparation process of the Y-shaped joint comprises the following steps:

1. annealing: mixing 10ul 100uM of joints E1 and E2, incubating at 95 ℃ for 5min, closing the machine, cooling for 1h, and taking out, so that slow cooling of two chains is ensured, and accurate annealing efficiency is improved;

2. extending and filling: the double-stranded sequence was extended and filled up by reacting at 37 ℃ for 1 hour using the following reagent mixture.

Table 15:

practice of	Volume (μ L)
		DNA	10
10 XNEB buffer 2	5
		25mM dNTP mix	2
Nuclease-free water	31
		Klenow exo-(5U/μL)	3
Total of	50

3. Y-shaped joint for extracting, extending and supplementing by ethanol precipitation method

(1) Water was added to 50ul extension to make up to 250ul, then 1.5 μ L Glycogen (20 μ g/. mu.L), 13 μ L3M NaAc, 600 μ L100% ethanol at-20 ℃ were added. Mixing, precipitating at-80 deg.C for at least 30min, and centrifuging at 14000rpm at 4 deg.C for 30 min. Carefully removing the supernatant, and leaving the bottom precipitate of the centrifuge tube;

(2) adding 1000 μ L70% ethanol stored at-20 deg.C, washing (using it as prepared), centrifuging at 14000rpm for 3min, and removing supernatant;

(3) washing with 70% ethanol once, centrifuging at 14000rpm for 3min, removing supernatant, drying precipitate (changing the precipitate from white to colorless after drying), adding 20 μ L of nuclease-free water to dissolve the precipitate, and incubating at 37 deg.C for 5 min;

(4) qubitration was performed on 1. mu.L of the solution, and a clean 0.5mL centrifuge tube was prepared, and the molar concentration was confirmed and the joints were connected.

4. Preparing a Y-shaped joint for library construction connection by enzyme digestion: the reaction is carried out for 1h at 37 ℃ according to the following reagent mixture ratio to complete the enzyme digestion reaction.

Table 16:

reagent	Volume (μ L)
		DNA	5.22
Nuclease-free water	16.28
		10 XNEB cutmarst buffer	2.5
HpyCH4III	1
		Total of	25

5. Performing gel cutting and recovery on a target band by using PAGE gel electrophoresis, and obtaining a finally used Y-shaped connector by using an ethanol precipitation method, wherein the mass range of the obtained connector is as follows: 56 ng-100 ng.

Table 17:

from the above description, it can be seen that the method of the present invention is a sequencing and library-building method with wide application and wide requirement for RNA quality, the initial amount can be as low as 50ng, and the method is suitable for eukaryotic transcriptome research in various life sciences and medical fields.

Compared with the prior art, the scheme of the application has at least the following advantages:

1) the method is an absolute quantitative library construction method suitable for degraded or low-RIN-value RNA samples, can obtain the same quality data including Q20, Q30, Mapping rate, Dup rate and the like by comparing normal or high-RIN-value RNA samples, and is suitable for library construction of different quality RNA samples extracted from large-scale nucleic acid in the industrial sequencing process.

2) The used joint is prepared by oligonucleotide annealing directly, other complex procedures are not needed, the joint usage amount of single library construction is only 2.5uM, the joint usage amount of one annealing synthesis is enough for ten thousand times, and the joint usage requirement of industrialized sequencing library construction is met;

3) simplifying the library building process, and completing library building only by three main operations; the operation of each link can be completely finished by an automatic mechanical arm, and the automatic industrial production scale is expected to be realized; in addition, the cost is calculated to be extremely low in price of a single reaction, and the price is similar to that of a common transcriptome library.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Sequence listing

<110> Tianjinnuo cereal-based bioinformatics science and technology Co., Ltd

<120> RNA library construction method

<130> PN141679TJNH

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(39)

<223> Joint 1

<220>

<221> misc_feature

<222> (34)..(38)

<223> nnnnnn represents UMI

<220>

<221> misc_feature

<222> (38)..(38)

<223> phosphorothioate linkage modification

<400> 1

acactctttc cctacacgac gctcttccga tctnnnnnt 39

<210> 2

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(37)

<223> Joint 2

<220>

<221> misc_feature

<222> (1)..(5)

<223> nnnnnn represents UMI

<220>

<221> misc_feature

<222> (1)..(1)

<223> phosphorylation modification

<400> 2

nnnnnagatc ggaagagcac acgtctgaac tccagtc 37

<210> 3

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(70)

<223> Joint E1

<220>

<221> misc_feature

<222> (22)..(33)

<223> UMI

<400> 3

gttacaattc ttctacagtc annnnnnnnn nnncaggaga tcggaagagc gtcgtgtagg 60

gaaagagtgt 70

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(34)

<223> Joint E2

<400> 4

gtgactggag ttcagacgtg tgctcttccg atct 34