CN116463405A - Chain-specific RNA sequencing method based on Tn5 transposon development - Google Patents
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a chain-specific RNA sequencing method based on Tn5 transposon development, which comprises the steps of extracting total RNA from cells, carrying out reverse transcription on the RNA to obtain cDNA/RNA hybrid chains, digesting the RNA chains to obtain cDNA with a single-chain structure, utilizing the characteristic that the Tn5 transposon can cut and mark the 5 'end of single-chain DNA, cutting fragmented cDNA chains by using the Tn5 transposon with a single joint, and inserting a specific DNA joint at the 5' end of the cDNA chains; the 3' end of the cDNA is connected with another specially designed double-stranded DNA joint containing special modification through T4DNA ligase; by using primers containing specific linker sequences, a strand-specific transcriptome library is constructed using PCR amplification. The invention uses the activity of the Tn5 transposon for cutting and marking DNA, develops a novel RNA strand specific sequencing method, and compared with the existing method, the library construction method has the advantages of convenient operation, cost saving and library preparation time saving; and reduces the amount of starting RNA required for high throughput sequencing, and can be applied to a plurality of RNA sequencing methods.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chain-specific RNA sequencing method based on Tn5 transposon development.
Background
RNA sequencing, i.e., transcriptome sequencing, is commonly used to analyze the expression of differential genes at the transcriptome level. With the development of the second generation sequencing technology, the RNA sequencing technology is also rapidly developed, and is applied to a plurality of life science fields.
The traditional RNA sequencing method is dUTP-seq, which incorporates dUTP into the substrate during cDNA second strand synthesis, and then uses UDG enzyme to digest the second strand template prior to PCR amplification, ensuring that the sequencing information is all from the first strand cDNA, thus preserving strand orientation of RNA sequencing. However, dUTP-seq library construction is complicated, the operation time is long, and the total amount of the initial RNA is large. Therefore, the development of a novel RNA strand specific sequencing method has practical application value. In view of the above problems, a solution is proposed below.
Disclosure of Invention
The invention aims to provide a chain-specific RNA sequencing method based on Tn5 transposon development, which has the function of firstly cutting and marking single-stranded DNA by the Tn5 transposon and applying the function to RNA sequencing, wherein the obtained library has chain specificity; the library construction method is convenient to operate; and the low amount of starting RNA required for this high throughput sequencing.
The technical aim of the invention is realized by the following technical scheme:
taking the mouse embryonic stem cell RNA transcriptome sequencing as an example, the whole flow of the high-throughput sequencing method of the strand-specific RNA is as follows:
(1) Culturing the mouse embryo stem cells, extracting RNA in the cells, and adding DNase I to remove residual DNA in the RNA.
(2) Reverse transcription of RNA to obtain cDNA. RNaseH and RNaseA are added to the cDNA system to digest RNA in the cDNA/RNA hybrid strand, and unreacted other RNA. The cDNA was denatured at high temperature, opening the potential secondary structure.
(3) The transposon Tn5-B, in which Tn5 transposase was assembled with linker adapter B to form a single linker, was used to cleave cDNA, and at the same time, linker adapter B (5 '-adapter B-cDNA) was added to the 5' -end of the cDNA, and DNA was purified using DNA purification beads.
(4) A double-stranded DNA adaptor adapter of a specific sequence was designed for ligation to the 3' end of the cDNA. One strand of AdapterA is 5' to an increased phosphorylation modification; the 3 'end of the other complementary strand of AdapterA contains 6 random DNA bases and an inverted dT base is added to the 3' end to prevent its misextension.
(5) The 3 '-end of the cDNA containing adapter B at the 5' -end was ligated to adapter A by T4DNA ligase to obtain a cDNA template with a double-end specific adaptor (5 '-adapter B-cDNA-Adapter A-3').
(6) PCR amplification was performed using primers matching AdaperA and AdaperB and cDNA containing double-ended adaptors as templates to obtain a strand-specific transcriptome library for high throughput sequencing.
Compared with the existing strand-specific sequencing technology dUTP-seq, the invention has the following advantages:
(1) The invention utilizes the function of cutting and marking single-stranded DNA by Tn5 transposon, and can directly add a joint at the 5' end of cDNA.
(2) The amount of starting RNA required can be as low as 30pg, corresponding to the amount of RNA of a single mouse embryonic stem cell.
(3) The library construction method is convenient to operate.
(4) The strand-specific sequencing provided by the invention can be applied to various RNA sequencing methods.
Drawings
FIG. 1 is a flow chart of an embodiment of the present invention;
FIG. 2 is a representation of the transcript strand-specific sequencing results of the present invention applied to 300ng of a large number of RNAs;
FIG. 3 shows the results of transcript strand-specific sequencing of 30pg trace amounts of the starting RNA according to the present invention.
Detailed Description
The following description is only of the preferred embodiments of the present invention, and the scope of the present invention should not be limited to the examples, but should be construed as falling within the scope of the present invention.
(one) intracellular RNA acquisition
(1) The method comprises the steps of removing culture medium from mouse embryonic stem cells obtained by cell culture, washing two sides by PBS, adding 0.3ml buffer RLT, gently blowing and mixing, adding 1/2 volume ethanol and mixing, transferring the mixed solution into an RNA adsorption column, and extracting total RNA of the cells by a column method.
(2) Mu.l of RNA was taken and 2. Mu.l of 10 Xbuffer, 1. Mu.l of DNaseI, 7. Mu. l H were added 2 O, reaction at 37℃for 1h, removal of DNA was contaminated, precipitated with 75% ethanol and purified to give RNA.
(II) reverse transcription of RNA to obtain cDNA Single-chain
(1) Using Thermo Fisher SuperScript TM IV kit (Thermo, cat# 18091050) reverse transcription kit, reverse transcription RNA, specific procedures are: mu.l of RNA was taken and a fixed amount of 10. Mu.l was added with 1. Mu.l of 50. Mu.M Oligo d (T) 20 and 1. Mu.l of 10mM dNTP, reacted at 65℃for 5 minutes, and immediately placed on ice for 2 minutes; subsequently, 4. Mu.l of 5 XSSIV buffer, 1. Mu.l of 100mM DTT, 1. Mu. l Ribonuclease Inhibitor, 1. Mu.l of SuperScript were added to the system TM Reverse Transcriptase 1. Mu.l of 100. Mu.g/ml Actinomycin D, 20. Mu.l of the system, were mixed thoroughly, reacted at 42℃for 90min, then at 50℃for 2min, at 42℃for 2min, reacted for 10 cycles, finally at 85℃for 5min, maintained at 16 ℃. At this time, RNA is reverse transcribed to synthesize first strand cDNA, which is the cDNA/RNA hybrid strand.
(2) 1. Mu.l of Exo I, 3. Mu.l of 10 Xbuffer, 6. Mu. l H were added to the cDNA/RNA hybrid system 2 O, reacting for 30min at 37 ℃, and inactivating for 15min at 80 ℃. The purpose of this step is to remove oligo d (T) 20 which has not been completely reacted in the reverse transcription system.
(3) 1. Mu.l of RNase A and 1. Mu.l of RNase H were added to the above-mentioned reacted system to digest RNA in the cDNA/RNA hybrid strand, and other RNA which had not been reacted. The cDNA was denatured at 98℃for 10min, and the potential secondary structure was opened to give a complete single-stranded cDNA.
(III) preparation of single-linker Tn5 transposon:
(1) Transposon single linker adapter b is a double stranded DNA structure formed by annealing and polymerizing two oligonucleotide strands. Mu.l of 100. Mu.M Tn5b (SEQ ID NO: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3') was taken together with an equivalent of 1. Mu.l of 100. Mu.M 5 '-terminal phosphorylated primer Tn5rev (SEQ ID NO: 5' - [ phos)]CTGTCTCTTATACACATCT-3’)，8μl H 2 Annealing and hybridizing the O mixed system, reacting for 5min at 95 ℃, then reducing the temperature to 25 ℃ at the speed of 0.1 ℃/min, and annealing to obtain 10 mu l of adaptor B;
(2) Mu.l of adaptor-B was incubated with 70. Mu.l of Tn5 transposase protein (concentration 0.5 mg/ml) at 25℃for 1h with mixing at room temperature to give a single adaptor transposase complex (Tn 5-B).
(IV) preparation of cDNA 3' -end adaptor adapter A:
(1) The 3' -end linker of cDNA is a double-stranded DNA structure formed by annealing and polymerizing two oligonucleotide chains. Mu.l of 100. Mu.M 5 '-terminal phosphorylated primer 5ph_Tn5a (sequence: 5' - [ phos)]CTGTCTCTTATACACATCTGACGCTGCCGACGA-3'), 2 μl of 100 μM of a specifically modified oligonucleotide Tn5a_N6_invent_dT complementary to Tn5a (sequence: 5' -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNN [ invent dT ]]-3’)，16μl H 2 Annealing and hybridizing the O mixed system, reacting for 5min at 95 ℃, then reducing the temperature to 25 ℃ at the speed of 0.1 ℃/min, and annealing to obtain 20 mu l of cDNA 3' -end connecting adaptor adapter containing modified double-chain structure;
(2) In the system of the 3' -end adaptor adapter A of the cDNA, 1. Mu.l of exonuclease EXO I, 3. Mu.l of 10 Xbuffer, 6. Mu. l H were added 2 O, 30. Mu.l of the system was used, incubated at 37℃for 2 hours, single strands of unreacted oligonucleotides were removed from the system, and the adaptor adapter was purified using 60. Mu.l of DNA purification beads.
(V) sequencing library construction:
(1) cDNA fragmentation: mu.l of cDNA of single-stranded structure was taken, 4. Mu.l of 5XDMF buffer (50% DMF,50mM Tris-HCl pH 7.5,10mM MgCl2) was added in sequence, 2. Mu.l of transposon Tn5-B was thoroughly mixed, reacted at 37℃for 5min, and the 5' -end of cDNA was cleaved and labeled. Stop buffer (1.5. Mu.l 0.5M EDTA, 1.8. Mu.l 10% SDS, and 0.5. Mu.l 20mg/ml Proteinase K) was added immediately after the reaction, reacted at 55℃for 30min, reacted at 70℃for 20min to terminate the reaction, and the DNA was purified using 21.4. Mu.l DNA purification beads. At this time, the cDNA is fragmented and simultaneously, an adapter B is added at the 5' end;
(2) 3' end adapter a connection: mu.l of purified cDNA containing adapterB was combined with 2. Mu.l of adapterA, 0.5. Mu. l T4DNA ligase, 2. Mu.l of 10 Xbuffer, 2.5. Mu.l of 40% PEG6000, 3. Mu. l H 2 O was thoroughly mixed and reacted at 37℃for 1 hour, and the DNA was purified using 16. Mu.l of DNA purification beads. At this time, the 3' -end of the cDNA was added with a linker adapter A, which was a double-ended-linker cDNA.
(3) Library amplification: PCR amplification was performed using cDNA with adaptor A/B attached to both ends as a template and primers containing specific index sequences. Because the template is single-stranded cDNA, the amplified product has chain specificity.
The upstream primer sequence is as follows: 5'-AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TCGTCGGCAGCGTCAGATGTGTAT-3' (XXXXXXXX, sequencing index); the downstream primer sequences were: 5'-CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGGAGATGTG-3' (XXXXXXXX, sequencing index). The PCR system is as follows: mu.l of template cDNA, 10. Mu.l of 2 Xhigh fidelity P CR enzyme, 1. Mu.l of upstream primer, 1. Mu.l of downstream primer, 3. Mu.l of water were mixed thoroughly; the PCR amplification procedure was: 5min at 72℃and 30s at 98 ℃;20 cycles: 98℃for 10s and 63℃for 10s; extension at 72℃for 1min,16℃hold.
And (six) sequencing and analyzing the library.
The technical problems, technical solutions and advantageous effects solved by the present invention have been further described in detail in the above-described embodiments, and it should be understood that the above-described embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of protection of the present invention.
Claims (8)
1. A method for sequencing chain-specific RNA based on Tn5 transposon development is characterized by comprising the following steps,
(1) Extracting RNA from cells, reversely transcribing the extracted RNA into cDNA/RNA hybrid chains, and then digesting the RNA chains by RNase to obtain cDNA chains;
(2) Cutting cDNA strand with single linker Tn5 transposon to fragment it while inserting specific DNA linker at 5' end of cDNA strand;
(3) The 3' end of the fragmented cDNA is connected with another specially designed double-stranded DNA joint through T4DNA ligase to prepare a cDNA chain with double-end specific joints;
(4) PCR amplification of cDNA strands with double-ended specific adaptors using primers containing specific DNA adaptor sequences yields a strand-specific transcriptome library useful for direct second-generation sequencing.
2. The method for strand-specific RNA sequencing based on the development of a Tn5 transposon according to claim 1, wherein the RNA is extracted from the cells in the step (1), the DNA remaining in the RNA is removed by DNase I, and the RNA strand and the unreacted RNA are removed by RNase after the cDNA is reverse transcribed in the step (1).
3. The method of claim 1, wherein the single linker in step (2) is transposon Tn5-B assembled with a linker adaptor B to form a single linker, the specific DNA linker inserted in step (2) is adaptor B, and the specific DNA linker is inserted to form a 5' -adaptor B-cDNA strand.
4. The method of claim 1, wherein the double-stranded DNA adaptor in step (3) is adaptor a, the adaptor a is attached to the 3 'end of the cDNA, one strand of the adaptor a is 5' modified by phosphorylation, the 3 'end of the other complementary strand of the adaptor a comprises 6 random DNA bases, and an inverted dT base is added to the 3' end of the adaptor a to prevent misextension.
5. The method of claim 1, wherein the T4DNA ligase in step (3) ligates the 3 '-end of the cDNA containing adapterB at the 5' -end with adapterA to obtain a cDNA template 5'-adapterB-cDNA-AdapterA-3' with a double-ended specific adaptor.
6. The method for strand-specific RNA sequencing based on development of Tn5 transposon according to claim 1, wherein the DNA adaptor of Tn5 transposon in step (2) is formed by high temperature annealing polymerization of two oligonucleotide strands, and the sequences are respectively Tn5b:5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' and Tn5rev:5'- [ phos ] CTGTCTCTTTATACACACACATCT-3'.
7. The method for sequencing a strand-specific RNA based on development of a Tn5 transposon according to claim 1, wherein the DNA adaptor inserted at the 3' end of the cDNA strand by the T4 ligase used in the step (3) is formed by high temperature annealing polymerization of two oligonucleotide strands, and the sequences are respectively 5ph_Tn5a:5'- [ phos ] CTGTCTCTTATACACATCTGACGCTGCCGACGA-3' and Tn5a_N6_index_dT: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNN [ invert dT ] -3'.
8. The method of claim 1, wherein the starting amount of RNA extracted in step (1) is as low as 30pg.
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