CN113025689A - Library construction method for modified small RNA and application thereof - Google Patents
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Abstract
The invention provides a library construction method of modified small RNA, which at least comprises the following steps: 1) removing amino acid residues at the tail end of the small RNA carrying the modification by using an amino acid removal reagent; 2) converting the 5-cap of the 5 'end of the modified small RNA into 5' P by using a first conversion agent; 3) converting the 5' -OH of the 5' end carrying the modified small RNA into 5' P, 3 ' P and 2 ', 3 ' -cP ends into 3 ' -OH by using a second conversion agent; 4) removing methylation modification of the modified small RNA by using a demethylating reagent; 5) connecting the modified small RNA to a sequencing joint; 6) reverse transcribing the modified small RNA into cDNA using a reverse transcription reagent comprising reverse transcriptase; 7) and purifying the cDNA, and performing PCR amplification to obtain a sequencing library carrying the modified small RNA. The invention provides a novel small RNA library construction method, which provides a powerful and sensitive analysis tool for enriching complex modified small RNAs carried in a living body.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to a library construction method of modified small RNA and application thereof.
Background
The non-coding RNA comprises a plurality of small RNAs such as siRNA, miRNA, piRNA and the like, which form a highly complex small RNA regulation network in cells and play an important regulation role in regulating nearly all events of the whole cell level such as ontogenesis, cell proliferation and differentiation, tumor occurrence and development, virus resistance and the like.
With the recent development of high-throughput RNA sequencing (RNA-seq) technology, we have recognized the rich RNA world in cells. Meanwhile, a large number of new types of small RNAs are found and reported in different species, including tsRNA (tRNA-derived small RNA), rsRNA (rRNA-derived small RNA), rasiRNA (repetitive related siRNA), hcRNA (heterochromatin RNA), and PASR/TASR (promoter terminator-related small RNA). Traditionally, RNA-seq is performed by adding a linker to the end of the RNA and reverse transcription using a primer complementary to the 3' linker. The method is suitable for transcripts with the structural characteristics of 5 '-phosphate groups and 3' -hydroxyl groups, and most of miRNA conforms to the structures, so that the sequencing technology for miRNA is relatively mature. However, for small RNAs carrying modifications at the end or inside, since they interfere with the ligation of the end linker or block reverse transcription process, a large number of small RNAs with modifications cannot be successfully pooled and thus are ignored as garbage fragments. Two articles in Nature Methods 2015 solved the technical problem of tRNA sequencing by removing tRNA modifications by enzymatic treatment prior to library preparation. At the same time, this method exploits a large number of tRNA fragments carrying methylated modified nucleosides. Many studies have shown that trnas can also be cleaved into smaller RNAs, even more abundant than micrornas. Research on tRNA-fragment shows that it is involved in cell proliferation, tumor formation, stem cell development, cross-generation inheritance, etc. These research results all indicate that there are abundant modified small RNAs in the living body, and that they store a lot of biological information closely related to the development of various physiological pathologies such as body development and tumor formation, and further research is urgently needed.
The establishment of a small RNA library construction method capable of capturing terminal and carrying modification inside is the first to overcome technical barriers for exploring and perfecting small RNA and deeply researching potential functions of the small RNA.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for library construction of small RNAs carrying modifications and uses thereof.
To achieve the above and other related objects, the first aspect of the present invention provides a method for constructing a library of small RNAs carrying modifications, comprising at least the steps of:
1) removing amino acid residues at the tail end of the small RNA carrying the modification by using an amino acid removal reagent;
2) converting the 5-cap of the 5 'end of the modified small RNA into 5' P by using a first conversion agent;
3) converting the 5' -OH of the 5' end carrying the modified small RNA into 5' P, 3 ' P and 2 ', 3 ' -cP ends into 3 ' -OH by using a second conversion agent;
4) removing methylation modification of the modified small RNA by using a demethylating reagent;
5) connecting the modified small RNA to a sequencing joint;
6) reverse transcribing the modified small RNA into cDNA using a reverse transcription reagent comprising reverse transcriptase;
7) and purifying the cDNA, and performing PCR amplification to obtain a sequencing library carrying the modified small RNA.
In a second aspect, the invention provides the use of the aforementioned method for library construction with modified small RNAs in the field of gene sequencing.
As described above, the present invention has the following advantageous effects: the invention provides a novel small RNA library construction method, which overcomes the obstruction of terminal modification and methylation in the reverse transcription process; the effective connection of the terminal joint is facilitated; the method has excellent persistence and fidelity; the preference of sequence abundance caused by PCR amplification is reduced, and the data analysis is real and reliable; provides a strong and sensitive analysis tool for the modified small RNA, and lays a solid research foundation for the function of the modified small RNA and the role play in the physiological and pathological processes and the like in the living body.
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FIG. 1 a: the invention is a simplified diagram of the principle and process of CPA-seq.
FIG. 1 b: annotated map of small RNA species corresponding to different banking methods in HEK293T cells.
FIG. 1c library construction analysis of small RNA of HEK293T cells by four small RNA library construction methods, NEBNext, QIAseq and TruSeq and CPA-seq.
FIG. 1d Venn diagram of the number of different types of small RNAs detectable by the four small RNA banking methods.
FIG. 2 a: t4PNK kinase responsive small RNA, distribution and species of T4PNK kinase responsive small RNA.
FIG. 2b distribution of T4PNK kinase-responsive small RNA, ribosomal RNA-derived small RNA responsive to T4PNK kinase in 5s ribosomal RNA, 18s ribosomal RNA and 28s ribosomal RNA.
FIG. 2c shows the sequencing length profile and Northern blot results (right) of T4PNK kinase-responsive small RNA, T4PNK kinase-responsive tsRNA with the highest expression (left), tsRNA (GluCTC).
FIG. 3 a: distribution of Small RNAs in response to Cap-Clip.
FIG. 3 b: distribution of snRNA-derived small RNAs in response to Cap-Clip.
FIG. 3 c: CPA-seq can detect small RNA with a cap structure at the 5' end of snRNA; northern hybridization of small RNAs treated with different enzymes using sequences complementary to RNU 15 'as probes, it was observed that Cap-Clip treatment successfully removed the 5' end Cap of RNU1 so that it could be cleaved by exonuclease XRN 1.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
All reagents described in the present invention are not limited to liquid form, as long as they can perform the corresponding functions.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
The library construction method of the present invention carrying modified small RNA is also referred to as CPA-seq or CPA.
As shown in fig. 1a, the library construction method for small RNA carrying modification provided in an embodiment of the present application at least includes the following steps:
1) removing amino acid residues at the tail end of the small RNA carrying the modification by using an amino acid removal reagent;
2) converting the 5-cap of the 5 'end of the modified small RNA into 5' P by using a first conversion agent;
3) converting the 5' -OH of the 5' end carrying the modified small RNA into 5' P, 3 ' P and 2 ', 3 ' -cP ends into 3 ' -OH by using a second conversion agent;
4) removing methylation modification of the modified small RNA by using a demethylating reagent;
5) connecting the modified small RNA to a sequencing joint;
6) reverse transcribing the modified small RNA into cDNA using a reverse transcription reagent comprising reverse transcriptase;
7) and purifying the cDNA, and performing PCR amplification to obtain a sequencing library carrying the modified small RNA.
Optionally, the modified small RNA is small RNA with a modified end and an internal modified end. The length of the modified small RNA carrying nucleotide is 15-40 nt.
Further, the methylation modification refers to a type of methylation modification of m1A, m1G and m 3C.
The modified small RNA is the small RNA carrying one or more modified genes selected from m1A, m1G, m3C and 5' -OH, 3 ' -P and 5' -cap. The detection efficiency of the small RNA carrying the modification by library construction and sequencing by the method provided by the invention is obviously improved.
The methylation modification can be located inside the small RNA.
Step 1), step 2) and step 3) facilitate efficient ligation of the end-linkers.
Optionally, in step 1), the amino acid removal reagent is selected from Tris-HCl buffer.
In one embodiment, the Tris-HCl buffer has a concentration of 0.1M and a pH of 9.0.
Further, in step 2), the first converting agent is selected from RNA5' acid pyrophosphate hydrolase (Cap-Clip).
Wherein the Cap-clip can convert RNA 5'-Cap into 5' -P.
In one embodiment, in step 3), the second conversion agent is selected from T4 polynucleotide kinase (T4 PNK). The second converting agent can convert 3 ' -P or dephosphorylation group at the 3 end of the small RNA into 3 ' -OH and 5' -OH at the 5' end into 5' P.
Further, in step 4), the methylation modification is selected from one or more of N1-methyladenosine (m1A), N3-methylcytosine (m3C) and N1-methylguanosine (m 1G). Overcomes the obstruction of methylation in the reverse transcription process.
In one embodiment, in step 4), the demethylating agent is selected from the group consisting of a mixture of E.coli-derived AlkB and its D135S mutant.
For the selection of AlkB and AlkB (D135S) mutants, reference is made to Zheng, G.et al.efficient and qualitative high-throughput tRNAsequencing. nat Methods 12,835-837, doi:10.1038/nmeth.3478 (2015).
Further, in step 5), a tag is included in the sequencing adaptor.
Optionally, the tag is umi (uniform Molecular identifier). The use of UMI greatly reduces the preference of sequence abundance due to PCR amplification, which in turn makes data analysis more realistic and reliable.
The sequencing adapters or UMIs are prior art and may be purchased or commissioned by a company.
In a preferred embodiment, the nucleotide sequence of the sequencing linker is as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, specifically:
5'-GUUCAGAGUUCUACAGUCCGACGAUC(N)(N)(N)(N)(N)(N)-3'；(SEQ ID NO：1)
this sequence is a 5-terminal linker, where (N) represents UMI (N ═ au C G equal ratio, i.e. the frequency of occurrence of each base at the corresponding position in the sequence of all sequencing linkers used for library construction is equal).
5'-P-(N)(N)(N)(N)(N)(N)(N)(N)(N)AGATCGGAAGAGCACACGTC-3ddC-3'；(SEQ ID NO：2)
This sequence is a 3-terminal linker, where (N) represents UMI (N ═ a T C G equal proportions).
Further, in step 6), the reverse transcriptase is selected from the group consisting of thermostable group II intron reverse transcriptase (TGIRT). Replacing the traditional reverse transcriptase (AMV or MMLV derived reverse transcriptase) with a thermostable group II intron reverse transcriptase (TGIRT) has superior persistence and fidelity in reverse transcription of structurally complex and re-modified RNA.
The aforementioned library construction method carrying modified small RNA can be used in the field of gene sequencing.
Example 1
Taking as an example the analysis in the human embryonic kidney cell line HEK293T cells:
CPA group:
step 1) isolation and purification of small RNA using mirVana mirRNAI (Life Technologies) kit,
step 2) taking 2 mu g of small RNA, and incubating the small RNA with Tris-HCl (pH 9.0) buffer and RNase inhibitor at 37 ℃ for 45 minutes to sufficiently remove amino acid residues at the tail end of the RNA;
step 3), purifying the small RNA by using ethanol;
step 4) incubating the mixture in 10 × Cap-Clip Acid Pyrophosphatase reaction buffer at 37 ℃ for 30 minutes to hydrolyze pyrophosphate bonds of RNA 5'-Cap to generate RNA with 5' -P terminal;
step 5) 20U T4PNK (NEB) is directly added into the reaction system of the step 4) and incubated for 30 minutes at 37 ℃ under the conditions of reaction buffer and 1mMATP (NEB) to repair the end of the small RNA and purify the RNA by phenol chloroform and ethanol; the reaction buffer composition was as follows: (70mM Tris-HCl, 10mM MgCl)2，5mM DTT，pH7.6@25℃)；
Step 6) 2-fold molar mass of AlkB and 4-fold molar mass of AlkB (D135S) in 300mM KCl,2mM MgCl2,10μM of(NH4)2Fe(SO4)2·6H2O,300 μ M2-ketoglutamate (2-KG),2mM L-ascorbic acid,50 μ g/ml BSA,50mM MESbuffer (pH 5.0) in reaction buffer at 25 ℃ for 1 hour to remove sufficiently the M1A, M3C and M1G methylation modifications of small RNAs;
step 7) adding 5mM EDTA to the reaction system to terminate the enzymatic reaction and purifying the RNA with phenol chloroform and ethanol;
step 8) RNA purified after the above three-step enzymatic reaction was ligated with T4RNA Ligase 2truncated KQ (NEB) and T4RNA Ligase 1(NEB) to a3 'linker and a5' linker comprising UMI, respectively (SEQ ID NO: 1, SEQ ID NO: 2) (ii) a
Step 9) incubating at 57 ℃ for 2h for reverse transcription under the mixing condition of NaCl, dNTPs, dithiothreitol and RNase inhibitor by using 200units of TGIRT-III;
step 10), carrying out gel running separation on RNA in 15% polyacrylamide modified gel containing 8M urea and carrying out gel cutting purification on corresponding RNA in a range of 15-50 nt;
step 11) preparation of NEBNext Ultra II Q5 Master Mix, SR Primer, Index (13-24) Primer and supplementation of nuclear-free water to a final volume of 50. mu.L reaction systems were subjected to 15 rounds of PCR amplification at 98 ℃ for 10s, 61 ℃ for 30s and 72 ℃ for 15s of extension. Performing electrophoresis on the PCR product in 6% polyacrylamide gel, and performing gel cutting and purification on the fragment with the size ranging from 140bp to 200 bp;
step 12) Illumina HiSeq X10 paired-end 2X 150bp sequencing was performed.
And step 13) carrying out annotation, differential analysis of different enzyme treatments and other related bioinformatics analysis on the small RNA species by using Bowtie (1.0.0) after Illumina sequencing.
PA group: the difference from the CPA group is that step 4) is not included, and the rest is the same;
group CA: the difference from the CPA group is that step 5) is not included, and the rest is the same;
and (3) CP group: the difference from the CPA group is that step 6) is not included, and the rest is the same;
group A: the difference from the CPA group is that step 4) and step 5) are not included, and the rest is the same;
and (3) group P: the difference from the CPA group is that step 4) and step 6) are not included, and the rest is the same;
group C: the difference from the CPA group is that step 5) and step 6) are not included, and the rest is the same;
untraded group: the difference from the CPA group is that step 2) -step 6) are not included, and the rest is the same;
NEBNext group: the Library was constructed according to the instructions in the NEBNext Multiplex Small RNA Library Prep Set for Illumina kit manufactured by NEB.
QIAseq group: the libraries were made according to the instructions in the QIAseq miRNA Library Kit (12) and the QIAseq miRNA 12Index IL (12) Kit, produced by QIAGEN.
TruSeqGroup (2):the library was constructed according to the instructions for the preparation of the kit according to the TruSeq small RNA library produced by Illumina.
The results in FIG. 1b show that the different enzymatic library construction methods have different capture capacity for each type of small RNA. CPA-seq revealed a greater variety of trnas, lncrnas, snornas, rrnas, mrnas, and other small RNAs derived from ncrnas, suggesting the presence of terminal or internal methylation modifications in non-miRNA small RNAs.
Comparing the CPA-seq with the results of sequencing small RNAs of HEK293T cells by three commercial small RNA banking methods NEBNext, QIAseq and TruSeq (collectively referred to as "NQT-seq") we found that different small RNA banking methods gave different results of analyzing the small RNAs (fig. 1 c). When analyzing the species of small RNAs we observed a large intersection between NQT-seq and the species of CPA-seq detected miRNA (FIG. 1 d). This is consistent with the conclusion that most miRNAs are known to terminate at the 5'-P and 3' -OH ends. However, CPA-seq revealed a greater variety of trnas, lncrnas, snornas, rrnas, mrnas and other small RNAs derived from ncrnas, indicating that the present invention is able to detect a greater abundance of small RNAs that were not previously captured.
To analyze small RNAs that respond to T4PNK kinase (these small RNAs may contain 5' -OH, 3 ' -P or 3 ' -cP), we compared the small RNAs detected in the CA and CPA groups. We found that most small RNAs (i.e. high detection in CPA group, low detection in CA group, fold change >30) responding to T4PNK kinase were derived from rRNA and tRNA derived fragments (fig. 2 a). Further, we found that most 5s ribosomal RNA-derived rRNA fragments could be captured without T4PNK kinase treatment. In contrast, most of the 18S and 28S ribosomal RNA-derived small RNAs required T4PNK kinase treatment to be captured by sequencing (fig. 2 b). And small 18s and 28s ribosomal RNA-derived RNAs are typically generated by cleavage of the RNA stem-loop region (fig. 2 b). Suggesting that small rRNA-derived RNAs may have different production mechanisms. In addition, small RNAs derived from tRNA in response to treatment with T4PNK kinase were derived primarily from the 5' end of the tRNA (fig. 2 c). Northern blot verification is carried out on the small RNA with the highest expression quantity from the 5' end of the GluCTC-tRNA, the result is quite consistent with the result analyzed by CPA-seq, and the invention is further proved to be capable of truly reflecting the expression level of the small RNA in cells.
Further, we compared the PA and CPA panels to explore the small RNA populations responding to Cap-Clip, and we found a large number of small RNAs derived from the 5' end of snRNA (FIG. 3 a). It is mainly derived from Sm snRNA, including RNU1 and RNVU1, and has been reported previously to have 5'-cap with m3G at the 5' end of this snRNA (fig. 3 b). Using a probe complementary to the 5 'sequence of U1 snRNA, we found that Cap-Clip uncapped U1 snRNA and its 5' fragment (U1-5 'f) ran somewhat faster in electrophoresis and was easily digested by XRN-1 (XRN-1 is a5' → 3 'exonuclease requiring 5' -P) (FIG. 3 c). This result confirmed the presence of 5' -cap-carrying small RNU 1-derived RNA in HEK293T cells.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
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Claims (10)
1. A method for constructing a library of small RNA carrying modifications, comprising at least the following steps:
1) removing amino acid residues at the tail end of the small RNA carrying the modification by using an amino acid removal reagent;
2) converting the 5-cap of the 5 'end of the modified small RNA into 5' P by using a first conversion agent;
3) converting the 5' -OH of the 5' end carrying the modified small RNA into 5' P, 3 ' P and 2 ', 3 ' -cP ends into 3 ' -OH by using a second conversion agent;
4) removing methylation modification of the modified small RNA by using a demethylating reagent;
5) connecting the modified small RNA to a sequencing joint;
6) reverse transcribing the modified small RNA into cDNA using a reverse transcription reagent comprising reverse transcriptase;
7) and purifying the cDNA, and performing PCR amplification to obtain a sequencing library carrying the modified small RNA.
2. The method of library construction of small RNA carrying modifications of claim 1, further comprising one or more of the following features:
a. the modified small RNA is small RNA with a modified end and a modified interior;
b. the length of the nucleotide carrying the modified small RNA is 15-40 nt;
c. in step 1), the amino acid removal reagent is selected from Tris-HCl buffer solution;
d. in step 2), the first conversion agent is selected from RNA5' acid pyrophosphate hydrolase.
3. The method for constructing a library of claim 1 wherein in step 3), said second transforming agent is capable of removing the phosphorylated group of 3 '-P or 2', 3 '-cP at 3' end of small RNA and converting the phosphorylated group of 5 '-OH at 5' end to 3 '-OH and 5' P.
4. The method for library construction of small RNA carrying modifications according to claim 1, wherein in step 3) the second transforming agent is selected from T4 polynucleotide kinase.
5. The method for constructing a library of small RNA carrying modifications according to claim 1 wherein in step 4) the methylation modification is selected from one or more of the group consisting of N1-methyladenosine, N3-methylcytosine and N1-methylguanosine methylation modifications.
6. The method for library construction with modified small RNA as claimed in claim 1, wherein in step 4), the demethylating agent is selected from the group consisting of E.coli-derived AlkB and a mixture of D135S mutants thereof.
7. The method for constructing a library of small RNA carrying modifications of claim 1, wherein in step 5) the sequencing adapter comprises a tag.
8. The method for constructing a library of small RNA carrying modifications of claim 7 wherein in step 5) the tag is UMI.
9. The method for constructing a library of claim 1 wherein in step 6) said reverse transcriptase is selected from the group consisting of thermostable group II intron reverse transcriptase.
10. Use of the method of library construction carrying modified small RNAs of claims 1-9 in the field of gene sequencing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023116490A1 (en) * | 2021-12-21 | 2023-06-29 | 中国科学院上海营养与健康研究所 | Novel method for detecting small rna and use thereof |
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