WO2021184146A1 - 一种待测样本rna的测序文库的构建方法 - Google Patents

一种待测样本rna的测序文库的构建方法 Download PDF

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WO2021184146A1
WO2021184146A1 PCT/CN2020/079434 CN2020079434W WO2021184146A1 WO 2021184146 A1 WO2021184146 A1 WO 2021184146A1 CN 2020079434 W CN2020079434 W CN 2020079434W WO 2021184146 A1 WO2021184146 A1 WO 2021184146A1
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rna
dna fragment
library
primer
cdna
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French (fr)
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夏军
陈健
刘萍
张薇婷
杨林
史千玉
张理菁
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深圳华大智造科技有限公司
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Priority to PCT/CN2020/079434 priority Critical patent/WO2021184146A1/zh
Priority to CN202080094602.1A priority patent/CN115003867A/zh
Publication of WO2021184146A1 publication Critical patent/WO2021184146A1/zh

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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • 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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  • the present invention belongs to the field of biotechnology, and specifically relates to a method for constructing a sequencing library of a sample RNA to be tested, in particular to a method for constructing a sequencing library of various RNAs (including mRNA, lncRNA, small RNA, cfRNA and tRNA) of the sample to be tested.
  • RNAs including mRNA, lncRNA, small RNA, cfRNA and tRNA
  • RNA sequencing also known as transcriptome sequencing
  • RNA-seq is currently the most widely used technology among high-throughput sequencing technologies. It performs high-throughput sequencing on RNA transcribed from tissues or cells at a specific time or in a functional state. .
  • RNA-seq can detect differences in the expression of all genes under various comparison conditions. For example, the difference between normal tissue and tumor tissue; the difference in gene expression before and after drug treatment; the difference in gene expression between different tissues at different developmental stages.
  • RNA-seq can study the transcription level of genes, as well as how to regulate gene expression at the transcription level, providing important technical support for the research of biological development, cell differentiation, tumorigenesis or disease pathogenesis.
  • the transcriptome refers to the sum of all RNA transcribed by a specific tissue or cell in a specific state, including mRNA and non-coding RNA (ncRNA); non-coding RNA mainly includes small RNA (small RNA), long-chain non-coding RNA (lncRNA) ), ribosomal RNA (rRNA) and transfer RNA (tRNA). Most of the total RNA mentioned is rRNA. Taking human cells or tissues as an example, about 95% of the total RNA mentioned is rRNA, 2-3% is mRNA, and 2-3% is lncRNA, tRNA, small RNA (such as miRNA, piRNA), etc. rRNA is very conserved throughout humans, and it is extremely stable in various human tissues and organs.
  • RNA-seq is mainly mRNA, followed by non-coding RNAs such as lncRNA and small RNA.
  • the length of mRNA and lncRNA are relatively long, generally greater than 200 nt; the length of small RNA is relatively short, about 20-30 nt.
  • RNA library construction The most important and basic step in RNA-seq is RNA library construction, and different methods of library construction are usually adopted for different RNAs that need to be studied.
  • RNAs such as mRNA or lncRNA
  • the common way to build a library is to fragment the RNA and perform reverse transcription to build a library of the obtained cDNA; the flow diagram of the traditional random primer reverse transcription method is shown in Figure 1.
  • the mRNA or rRNA is removed The total RNA (total RNA) is fragmented, and then a random primer is used for reverse transcription to synthesize one-strand cDNA, followed by two-strand cDNA synthesis, followed by end repair plus A, linker ligation, and PCR amplification to complete the RNA library construction.
  • the traditional random primer reverse transcription method can only build a library of long fragments of RNA (such as mRNA and lncRNA), but cannot build a small RNA library.
  • Use oligo dT method to selectively reverse transcription of RNA with polyadenine (polyA) tail, and build a library of the obtained cDNA the flow chart of the traditional Smart-seq library building method is shown in the figure 2.
  • Use reverse transcription primers with oligo dT to reverse transcription of mRNA, total RNA or total RNA from which rRNA has been removed.
  • the MMLV reverse transcriptase used can be added to the 5'end of the cDNA after reverse transcription of the full-length RNA 3 C bases, and then use TSO (Template switch oligo) primers for template conversion.
  • TSO primer 3' consists of 3 guanine ribonucleotides (rGrGrG) and 5'same primers.
  • the universal primers are used to amplify the full-length double-stranded cDNA, followed by interruption and end repair plus A, adaptor ligation and PCR to obtain an RNA library.
  • the traditional Smart-seq library building method can only build libraries for RNAs with polyA tails (such as mRNA and some lncRNAs), and cannot build libraries for lncRNAs and small RNAs without polyA tails.
  • reverse transcription is carried out by adding a single link head, and the amplified cDNA is then built into a library: the flow diagram of the traditional single link head connection small RNA library construction method is shown in Figure 3.
  • the total RNA or total RNA is connected to the 3'adaptor, then the 3'adaptor is digested, and then the 5'adaptor is connected, and then reverse transcription and amplification are performed.
  • the amplification primers include the sequencing adaptor to obtain double-stranded cDNA, and then proceed Fragment selection, you can get a small RNA library.
  • the traditional small RNA library building method with single link head connection can only build a library for short fragments of samll RNA, but cannot build a library for long fragments of RNA (such as mRNA and lncRNA); and the process of library construction usually requires complicated Fragment recovery, such as gel cutting and purification.
  • Fragment recovery such as gel cutting and purification.
  • RNA is at least one of mRNA, lncRNA, small RNA (including miRNA, siRNA, piRNA, etc.), cfRNA and tRNA.
  • the present invention first protects the construction method of RNA sequencing library, which may include the following steps:
  • step (1) adding poly(A) to the 3'end of the RNA of the sample to be tested is achieved by Poly(A) polymerase.
  • the reaction system of adding poly(A) to the 3'end of the RNA of the sample to be tested includes Poly(A) polymerase, Poly(A) polymerase reaction buffer, dATP and RNase inhibitor.
  • the reaction conditions for adding poly(A) to the 3'end of the RNA of the sample to be tested are: 35-39°C (such as 35-37°C, 37-39°C, 35°C, 37°C or 39°C). °C) incubate for 20-40min (such as 20-30min, 30-40min, 20min, 30min or 40min).
  • the Poly(A) polymerase is preferably E. coli Poly(A) Polymerase.
  • the primer for reverse transcription can include DNA fragment A and DNA fragment C from the 5'end to the 3'end.
  • the sequence length of DNA fragment A and DNA fragment C is different. It can be selected arbitrarily in the case of affecting its function.
  • the DNA fragment A can be any sequence of 15-20 bp (such as 15-18 bp, 18-20 bp, 15 bp, 18 bp or 20 bp).
  • the DNA fragment C may include 20-30 (such as 20-25, 25-30, 20, 25 or 30) T and 1 V from the 5'end to the 3'end.
  • the DNA fragment C may be composed of 20-30 (such as 20-25, 25-30, 20, 25 or 30) T and 1 V.
  • the DNA fragment C may also include 1 N, which is located at the 3'end of the V.
  • the DNA fragment C includes 20-30 (such as 20-25, 25-30, 20, 25 or 30) T, 1 V and 1 N from the 5'end to the 3'end.
  • the DNA fragment C may be composed of 20-30 (such as 20-25, 25-30, 20, 25 or 30) T, 1 V and 1 N.
  • the N is any one of A, T, C, and G
  • V is any one of A, C, and G.
  • the primer (from the 5'end to the 3'end) for reverse transcription can be composed of the DNA fragment A and the DNA fragment C.
  • the primer for reverse transcription may also include DNA fragment B, which is located in the middle of DNA fragment A and fragment C.
  • DNA fragment B can be a barcode sequence of 4-16 bp (such as 4-8 bp, 8-12 bp, 12-16 bp, 4 bp, 8 bp, 12 bp or 16 bp), which is used to distinguish different samples to be tested.
  • the primer for reverse transcription includes the DNA fragment A, the DNA fragment B, and the DNA fragment C from the 5'end to the 3'end.
  • the primer (from the 5'end to the 3'end) for reverse transcription can be composed of the DNA fragment A, the DNA fragment B, and the DNA fragment C.
  • the universal primer sequence may be a sequencing linker sequence.
  • the reverse transcriptase will add 3 Cs to the 3'end of the cDNA to obtain a cDNA labeled with C at the 3'end.
  • step (2) is completed and before step (3) is performed, a purification step may also be included.
  • the 3'end of the cDNA after terminal transferase treatment can be composed of 8-17 (such as 8-13, 13-17, 8, 13, or 17) B, and B is Any of T, C, and G.
  • the reaction system for terminal transferase treatment may include terminal transferase, terminal transferase reaction buffer and CoCl2.
  • the reaction system for terminal transferase treatment may also include dCTP, dGTP or dTTP.
  • the reaction conditions for terminal transferase treatment are: 35-39°C (such as 35-37°C, 37-39°C, 35°C, 37°C or 39°C) and incubate for 1-2h (such as 1- 1.5h, 1.5-2h, 1h, 1.5h or 2h).
  • the two-strand synthesis preferably includes a PCR amplification reaction.
  • the primer 1 for PCR amplification may include a DNA fragment a, a DNA fragment b, and a V from the 5'end to the 3'end; the DNA fragment a may be the reverse of the 3'end of the cDNA.
  • DNA fragment b can be GGG; primer 2 for PCR amplification can be a part of the 5'end of the primer for reverse transcription.
  • the primer 1 for PCR amplification can be composed of the DNA fragment a, the DNA fragment b, and one V from the 5'end to the 3'end.
  • any of the primers 1 for PCR amplification described above may also include 1 N, which is located at the 3'end of the V.
  • the primer 1 for PCR amplification described above includes the DNA fragment a, the DNA fragment b, 1 V and 1 N from the 5'end to the 3'end.
  • the primer 1 for PCR amplification described above can be composed of the DNA fragment a, the DNA fragment b, 1 V and 1 N from the 5'end to the 3'end.
  • the N is any one of A, T, C, and G
  • V is any one of A, C, and G.
  • the primer 2 for PCR amplification may be a primer for reverse transcription or the DNA fragment A or a part of the 5'end of the DNA fragment A.
  • V may be any of A, C, and G.
  • N can be any of A, T, C, and G.
  • the primer pair for PCR amplification is composed of the primer 1 and the primer 2.
  • step (4) is completed and before step (5) is performed, a purification step may also be included.
  • the nucleotide sequence of the primer for reverse transcription may specifically be 5'-CGACATGGCTACGATCCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'.
  • the nucleotide sequence of primer 1 for PCR amplification may specifically be 5'-GGGGGGGGGGGGGGGGGGVN-3', where N is any one of A, T, C, and G, and V is any one of A, C, and G kind.
  • the nucleotide sequence of primer 2 for PCR amplification may specifically be 5'-CGACATGGCTACGATCCGACTT-3'.
  • any of the above construction methods may also include the step of removing rRNA; the removal of rRNA can be performed before adding poly(A) to the 3'end in step (1), or after obtaining double-stranded cDNA in step (4).
  • the rRNA removal can be accomplished by rRNA removal reagents.
  • obtaining the RNA sequencing library further includes the following steps:
  • step (5) after step (c1) is completed and before step (c2) is performed, a purification step may also be included.
  • step (5) after completing step (c3) and before proceeding to step (c4), a purification step may also be included.
  • step (5) after step (c4) is completed and before step (c5) is performed, a purification step may also be included.
  • step (5) after step (c4) is completed and before step (c5) is performed, a nucleic acid quantification step may also be included. After nucleic acid quantification is completed, a purification step may also be included.
  • the purpose of single-stranded circularization can be to circularize single-stranded linear DNA into a single-stranded circular DNA library.
  • the purpose of enzymatic digestion may be to digest single-stranded linear DNA and/or double-stranded linear DNA that has not been circularized.
  • a purification step may also be included.
  • a nucleic acid quantification step may also be included.
  • sequencing may also be included after the library construction is completed.
  • the sequencing can be performed using a high-throughput sequencer (such as MGISEQ-2000 sequencer).
  • any of the above-mentioned purification can be achieved by agecourt AMPure XP magnetic beads.
  • the purified nucleic acid is soluble in TE buffer.
  • the sample to be tested can be any biological sample such as cells, tissues, or body fluids.
  • the tissue may specifically be mouse brain tissue.
  • the present invention also protects the application of any one of the above-mentioned construction methods in the analysis of the RNA of the sample to be tested.
  • the RNA may be at least one of mRNA, lncRNA, small RNA, cfRNA and tRNA.
  • the small RNA is at least one of miRNA, siRNA and piRNA.
  • the present invention also protects an RNA library construction kit, which may include Poly(A) polymerase, dATP, terminal transferase, reverse transcriptase, any one of the above-mentioned primers for reverse transcription, and any one of the above-mentioned The primer 1 for PCR amplification and/or the primer 2 for PCR amplification described above.
  • the kit can specifically be made of Poly(A) polymerase, dATP, terminal transferase, reverse transcriptase, any one of the above-mentioned primers for reverse transcription, any one of the above-mentioned primers 1 for PCR amplification, and the above Any one of the primers 2 for PCR amplification is composed.
  • kits may also include any one of the above rRNA removal reagents and/or library building reagents.
  • kits can specifically be made of Poly(A) polymerase, dATP, terminal transferase, reverse transcriptase, any one of the above-mentioned primers for reverse transcription, and any one of the above-mentioned primers for PCR amplification 1.
  • the present invention also protects the application of any of the above kits, which can be T1) or T2):
  • the sample to be tested may be any biological sample such as cells, tissues, or body fluids.
  • the tissue may specifically be mouse brain tissue.
  • RNA may be at least one of mRNA, lncRNA, small RNA, cfRNA, and tRNA.
  • Any of the aforementioned small RNAs may be at least one of miRNA, siRNA, and piRNA.
  • the present invention first uses an enzyme with the function of adding poly(A) to RNA to add poly(A) to RNA, then reverse transcription is carried out with oligo dT primers and reverse transcriptase, and then terminal transferase is used to add 3'to the cDNA.
  • the method of adding a fixed sequence to the end as a primer anchor point can simultaneously build a library of a variety of RNAs, including long fragments of mRNA and lncRNA with poly(A), long fragments of lncRNA and short fragments of different poly(A) Small RNA without poly(A).
  • the present invention can construct a library of multiple RNAs (including mRNA, lncRNA, small RNA, cfRNA and tRNA) at the same time.
  • the constructed library includes mRNA library, lncRNA library, small RNA library, cfRNA library and tRNA library at the same time.
  • There is no need for special fragment recovery methods to recover small RNA libraries so that you only need to build a library once for an RNA sample, and you can obtain information about multiple RNAs at the same time. It is necessary to divide a piece of RNA into several pieces and construct a library of different RNAs separately, thereby improving the efficiency and simplicity of RNA library construction.
  • the invention has important application value.
  • Figure 1 is a schematic diagram of the process of constructing a sequencing library using a traditional random primer reverse transcription method.
  • Figure 2 is a schematic diagram of the process of constructing a sequencing library using the traditional Smart-seq library construction method.
  • Figure 3 is a schematic diagram of the process of constructing a sequencing library using a traditional single-linked small RNA library construction method.
  • Fig. 4 is a schematic flow chart of constructing a sequencing library based on the method of constructing a library based on RNA plus polyA tail and terminal transferase (that is, the method provided by the present invention).
  • test materials used in the following examples are all purchased from conventional biochemical reagent stores.
  • the specifications of the PCR tubes are all 200 ⁇ L.
  • the specifications of the centrifuge tube are 1.5mL.
  • the mouse involved in the following examples is specifically C57BL/6J.
  • the MGIEasy rRNA Removal Kit is a product of Shenzhen Huada Intelligent Manufacturing Technology Co., Ltd., the product number is 1000005953.
  • Nuclease Free Water is a product of AMBION Company, the article number is AM9932.
  • Example 1 Construction of a mouse brain tissue RNA sequencing library
  • the inventor of the present invention has established a method for constructing an RNA sequencing library of mouse brain tissue through a large number of experiments. Specific steps are as follows:
  • RNA-removed RNA Take 200ng of total RNA from mouse brain tissue, use MGIEasy rRNA removal kit to remove rRNA in total RNA (specifically follow the instructions of the kit), and finally dissolve it with 10 ⁇ L of water to obtain mouse rRNA-removed RNA.
  • the rRNA removed from mice contains mRNA with poly(A) and RNA without poly(A), and RNA without poly(A) such as lncRNA, tRNA, miRNA, and piRNA.
  • E. coli Poly(A) Polymerase is a product of NEB Company, the product number is M0276S. 10 ⁇ E.coli Poly(A) Polymerase Reaction Buffer is a component of E. coli Poly(A) Polymerase.
  • step 2 After completing step 1, take the PCR tube, add the rRNA-removed RNA from the mouse obtained in step 1, and mix well.
  • step 3 After completing step 2, put the PCR tube in a PCR machine and incubate at 37°C for 30 minutes; then add 3 ⁇ L of 0.5M EDTA aqueous solution to stop the reaction, and obtain mouse RNA with poly(A) added.
  • RT primer The nucleotide sequence of RT primer is:
  • step 2 After completing step 1, take the PCR tube (which contains mouse plus poly(A) RNA) that has been completed in step 2 and 3, add the mixed reagent 1 and mix well.
  • step 2 After completing step 2, put the PCR tube in a PCR machine, incubate at 37°C for 30 minutes, at 45°C for 10 minutes, at 55°C for 10 minutes, and at 65°C for 10 minutes.
  • Reverse Transcriptase (Alpha Reverse Transcriptase) will add 3 Cs to the 3'end of the reverse transcription product to obtain a cDNA labeled with C at the 3'end.
  • Terminal Transferase is a product of NEB Company, the article number is M0315S. 10 ⁇ Terminal Transferase Reaction Buffer is a component in Terminal Transferase.
  • step 2 After completing step 1, take the PCR tube, add the reverse transcription product recovered in step 3, 4, and mix.
  • step 3 After completing step 2, put the PCR tube in a PCR machine and incubate at 37°C for 1.5 hours.
  • Terminal Transferase (Terminal Transferase) will add several Cs after the C at the 3'end of the product.
  • the nucleotide sequence of cDNA PCR Primer-F is 5'-GGGGGGGGGGGGGGGGGGVN-3', where N is any one of A, T, C, and G, and V is any one of A, C, and G.
  • the nucleotide sequence of cDNA PCR Primer-R is 5'-CGACATGGCTACGATCCGACTT-3'.
  • step 2 After completing step 1, add the mixed reagent 2 to the reaction system after completing step 4 in 3, and mix well.
  • step 2 After completing step 2, put the PCR tube in a PCR machine to perform PCR amplification to obtain a cDNA amplification product.
  • the reaction procedure is: 95°C 2min; 95°C 10s, 60°C 20s, 72°C 3min, 15 cycles; 72°C 5min; 4°C hold.
  • step 5 Transfer the cDNA amplification product obtained in step 5, step 3, to a centrifuge tube, and then add 150 ⁇ L agencourt AMPure XP magnetic beads for purification.
  • the purified product is dissolved in 23 ⁇ L TE buffer, and 21 ⁇ L of purified cDNA is recovered.
  • step 2 After completing step 1, take 1 ⁇ L of purified cDNA and use Qubit dsDNA HS Assay kit (Invitrogene) to determine the concentration.
  • MGIEasy universal DNA library preparation reagent kit is a product of MGI Company, the catalog number is 1000006985.
  • the fragmentation reaction solution is 4 ⁇ L, which is a mixture of 2 ⁇ L Fragmentase and 2 ⁇ L 10 ⁇ Fragmentase buffer.
  • the end repair reaction solution is 10 ⁇ L, which is a mixture of 7.1 ⁇ L ERAT Buffer and 2.9 ⁇ L ERAT Enzyme Mix.
  • the adapter connection reaction solution is 30 ⁇ L, which is a mixture of 23.4 ⁇ L Ligation Buffer, 5 ⁇ L Adapter and 1.6 ⁇ L DNA Ligase.
  • the PCR reaction mixture is 30 ⁇ L, which consists of 25 ⁇ L PCR Enzyme Mix and 5 ⁇ L PCR Primer Mix.
  • the single-stranded cyclization reaction solution is 12.1 ⁇ L, which is a mixture of 11.6 ⁇ L Splint Buffer and 0.5 ⁇ L DNA Rapid Ligase.
  • the enzyme digestion reaction solution is 4 ⁇ L, which is a mixture of 1.4 ⁇ L Digestion Buffer and 2.6 ⁇ L Digestion Enzyme.
  • step 2 place the PCR tube in a PCR machine (the hot lid is set to 75°C), and incubate at 37°C for 10 min; after the reaction is over, add TE buffer to a total volume of 50 ⁇ L, mix well and centrifuge for the reaction. The liquid is collected to the bottom of the tube.
  • step 1 and step 2 The purpose of step 1 and step 2 is to fragment the purified cDNA.
  • step 3 After completing step 2, take the PCR tube and add 75 ⁇ L agencourt AMPure XP magnetic beads for purification, the purified product is dissolved in 42 ⁇ L TE buffer, and 40 ⁇ L fragmented product is recovered.
  • step 3 take the PCR tube, add 10 ⁇ L end repair reaction solution and 40 ⁇ L fragmentation product, and mix.
  • step 4 place the PCR tube in a PCR machine (the hot lid is set to 75°C), incubate at 37°C for 30 minutes, incubate at 65°C for 15 minutes, and hold at 4°C to obtain the end repair product.
  • the hot lid is set to 75°C
  • step 4 and step 5 are to add "A" to the end repair.
  • step 5 take the PCR tube, add 30 ⁇ L of the adapter connection reaction solution, and mix well.
  • step 6 place the PCR tube in a PCR machine (the hot lid is set to 75°C), incubate at 23°C for 60 minutes, and keep it at 4°C.
  • step 6 and step 7 are to connect the joints.
  • step 7 take the PCR tube, add TE buffer to a total volume of 100 ⁇ L, and then use 100 ⁇ L of agencourt AMPure XP magnetic beads for purification, the purified product is dissolved in 22 ⁇ L of TE buffer, and 20 ⁇ L of linker connection product is recovered.
  • step 8 After completing step 8, take the PCR tube, add 20 ⁇ L of adapter ligation product and 30 ⁇ L of PCR reaction mixture, and mix well.
  • step 9 put the PCR tube in a PCR machine and perform PCR amplification to obtain PCR amplification products.
  • reaction procedure is: 95°C for 3min; 98°C for 20s, 60°C for 20s, 72°C for 30s, 13 cycles; 72°C for 5min; 4°C hold.
  • step 11 After completing step 10, take the PCR amplification product and add 75 ⁇ L agencourt AMPure XP magnetic beads for purification. The purified product is dissolved in 32 ⁇ L TE buffer, and 30 ⁇ L of the PCR purified product is recovered.
  • dsDNA HS Assay Kit product of Invitrogen, product number Q32851
  • Quant-iTTM The dsDNA Assay Kit (product of Invitrogen, catalog number P11496) and other double-stranded DNA fluorescence quantification kits quantify the PCR purified product recovered in step 11.
  • step 13 place the PCR tube in a PCR machine at 95°C for 5 minutes. After the reaction is over, immediately transfer the PCR tube to ice, leave it to stand for 2 minutes, and then centrifuge it immediately.
  • step 14 After completing step 14, take the PCR tube, add 12.1 ⁇ L of single-stranded cyclization reaction solution, vortex and shake 3 times, 3 seconds each time, and collect the reaction solution to the bottom of the tube by instant centrifugation.
  • step 16 After completing step 15, put the PCR tube in a PCR machine and react at 37°C for 30 minutes. After the reaction is over, centrifuge it briefly and transfer the PCR tube to ice.
  • step 15 and step 16 are to circularize single-stranded linear DNA into a single-stranded circular DNA library.
  • step 16 After completing step 16, take the PCR tube, add 4 ⁇ L of enzyme digestion reaction solution, vortex and shake 3 times, 3 seconds each time, and collect the reaction solution to the bottom of the tube by instant centrifugation.
  • step 17 put the PCR tube in a PCR machine and react at 37°C for 30 min. After the reaction is over, add 7.5 ⁇ L Digestion Stop Buffer to the PCR tube, vortex and shake 3 times, 3 seconds each time, and centrifuge to remove The reaction solution is collected to the bottom of the tube, and all the reaction solution is transferred to a new centrifuge tube.
  • the purpose of performing steps 17 and 18 is to digest single-stranded linear DNA and double-stranded linear DNA that have not been circularized.
  • the addition of Digestion Stop Buffer is to stop the action of digestive enzymes.
  • step 18 After completing step 18, take the centrifuge tube and add 170 ⁇ L agencourt AMPure XP magnetic beads for purification, the purified product is dissolved in 22 ⁇ L TE buffer, and 20 ⁇ L of the digestion product is recovered.
  • step 19 After completing step 19, take the digested product by restriction enzyme and use ssDNA Assay Kit fluorescence quantification kit for quantification.
  • the digested product is the mouse brain tissue RNA sequencing library.
  • MGISEQ-2000 high-throughput sequencing reagent set SE50
  • SE50 high-throughput sequencing reagent set
  • mouse brain tissue RNA sequencing library contained the nucleotide sequences of miRNA, tRNA, piRNA, mRNA and lncRNA.
  • step 2 quantify small RNA (such as miRNA, piRNA), mRNA and lncRNA through TPM (Transcripts Per Million reads, the number of corresponding transcripts in 1 million detected sequences).
  • small RNA such as miRNA, piRNA
  • mRNA and lncRNA Transcripts Per Million reads, the number of corresponding transcripts in 1 million detected sequences.
  • mice brain tissue RNA sequencing library prepared by the above steps contained 17,794 mRNA, 526 lncRNA, and 1105 small RNA.
  • step one can also be performed after step six is completed and before step seven is performed, at this time step one is step A.
  • the removal of rRNA can be carried out using the MGIEasy rRNA removal kit.
  • Step A Obtain the total RNA of mouse brain tissue
  • the total RNA of mouse brain tissue contains mRNA with poly(A) and RNA without poly(A), such as rRNA, lncRNA, tRNA, miRNA, and piRNA.
  • the method provided by the present invention is a library construction technology for simultaneously constructing a library of multiple RNAs.
  • the core is to add poly(A) to the RNA without poly(A), so that all linear RNAs have There is poly(A), and the primers with oligo dT are hybridized with poly(A) and then amplified by reverse transcription to obtain a strand of cDNA. Then use terminal transferase to add consecutive nucleotides to the 3'end of the cDNA as cDNA amplification primers are used to amplify cDNA, and then build a library.
  • Figure 4 shows the schematic flow diagram of the RNA sequencing library of the sample to be tested.
  • the present invention can construct a library of multiple RNAs (including mRNA, lncRNA, small RNA, cfRNA and tRNA) at the same time.
  • the constructed library includes mRNA library, lncRNA library, small RNA library, cfRNA library and tRNA library at the same time.
  • the invention improves the efficiency and convenience of RNA library construction, and has important application value.

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Abstract

本发明公开了一种待测样本RNA的测序文库的构建方法。该方法先使用Poly(A)聚合酶给RNA加上poly(A)，然后逆转录，之后使用末端转移酶给cDNA的3'端加上固定序列作为引物锚定位点，可以同时对多种RNA(包括mRNA、lncRNA、small RNA、cfRNA和tRNA)进行建库，构建的文库中同时包括mRNA文库、lncRNA文库、small RNA文库、cfRNA文库和tRNA文库，并且过程中不需要特殊的片段回收方法去回收small RNA文库，使得对一份RNA样本只需要建一次库，就可以同时得到多种RNA的信息，从而提高的RNA建库效率和简便性。本发明具有重要的应用价值。

Description

一种待测样本RNA的测序文库的构建方法 技术领域
本发明属于生物技术领域，具体涉及一种待测样本RNA的测序文库的构建方法，尤其涉及待测样本多种RNA(包括mRNA、lncRNA、small RNA、cfRNA和tRNA)的测序文库的构建方法。
背景技术
RNA测序(RNA-seq)又称转录组测序，是目前高通量测序技术中被用得最广的一种技术，对组织或细胞在特定时间或功能状态下转录的RNA进行高通量测序。RNA-seq可以在各种比较条件下，检测所有基因表达情况的差异。例如正常组织和肿瘤组织之间的差异；药物治疗前后基因表达的差异；不同的发育阶段，不同的组织之间的基因表达差异。RNA-seq可以研究基因的转录水平，也可以研究在转录水平如何调控基因表达，为生物的发育、细胞分化、肿瘤发生或疾病发病机制的研究提供了重要技术支持。
转录组是指特定组织或细胞在特定状态下转录出来的所有RNA的总和，其中包括mRNA和非编码RNA(ncRNA)；非编码RNA主要包括小RNA(small RNA)、长链非编码RNA(lncRNA)、核糖体RNA(rRNA)和转运RNA(tRNA)。在通常抽提到的总RNA中，绝大部分都是rRNA。以人类的细胞或组织为例，一般抽提到的总RNA当中，95％左右为rRNA，2-3％为mRNA，2-3％为lncRNA、tRNA、small RNA(如miRNA、piRNA)等。rRNA在整个人类当中都是非常保守的，而且在人的各个组织、器官当中也是极度稳定的。因此，一般RNA-seq的研究对象主要是mRNA，其次是lncRNA和small RNA这些非编码RNA。mRNA和lncRNA的长度都比较长，一般大于200nt；small RNA的长度则比较短，在20-30nt左右。
RNA-seq中最主要也是最基本的步骤RNA文库构建，通常针对需要研究的RNA的不同采取不同的建库方式。针对mRNA或lncRNA这些长链RNA，常见建库方式是将RNA片段化后进行逆转录，对得到的cDNA进行建库；传统随机引物逆转录方法的流程示意图见图1，对mRNA或去除了rRNA的total RNA(全RNA)进行片段化，然后采用随机引物进行逆转录合成一链cDNA，之后进行二链cDNA合成，随后进行末端修复加A，接头连接，PCR扩增后完成RNA文库构建。传统随机引物逆转录方法只能对长片段的RNA(例如mRNA和 lncRNA)进行建库，不能构建small RNA文库。对具有多聚腺嘌呤(polyA)尾的RNA采用胸腺嘧啶寡核苷酸(oligo dT)的方法选择性逆转录，对得到的cDNA进行建库：传统Smart-seq建库方法的流程示意图见图2，采用带有oligo dT的逆转录引物对mRNA、去除rRNA的total RNA或total RNA进行逆转录，使用的MMLV逆转录酶可以在完成全长RNA逆转录后，在cDNA的5’末端加上3个C碱基，然后使用TSO(Template switch oligo)引物进行模板转换，TSO引物3’由3个鸟嘌呤核糖核苷酸(rGrGrG)和5’的同用引物组成，完成逆转录后，使用通用引物扩增得到全长的双链cDNA，之后进行打断和末端修复加A、接头连接与PCR，得到RNA文库。传统Smart-seq建库方法只能对有polyA尾的RNA(例如mRNA和部分lncRNA)进行建库，不能对没有polyA尾的lncRNA和small RNA进行建库。对于片段长度较短的small RNA采用加单链接头的形式进行逆转录，扩增得到的cDNA之后建库：传统单链接头连接的small RNA建库方法的流程示意图见图3，对去除rRNA的total RNA或total RNA进行3’接头的连接，然后消化3’接头后，再连接上5’接头，之后进行逆转录和扩增，扩增用的引物包含测序接头，得到双链cDNA，再进行片段选择，就可以得到small RNA文库。传统单链接头连接的small RNA建库方法只能对短片段的samll RNA进行建库，不能对长片段的RNA(例如mRNA和lncRNA)进行建库；而且建库过程中，通常需要进行复杂的片段回收，如切胶纯化。目前，还没有一种同时对多种RNA进行文库构建的方法。
发明公开
本发明的目的是构建RNA测序文库，从而分析RNA；RNA为mRNA、lncRNA、small RNA(包括miRNA、siRNA、piRNA等)、cfRNA和tRNA中的至少一种。
本发明首先保护RNA测序文库的构建方法，可包括如下步骤：
(1)取待测样本的RNA，3’末端加poly(A)；
(2)反转录，获得cDNA；
(3)末端转移酶处理；
(4)二链合成，获得双链cDNA；
(5)获得RNA测序文库。
所述步骤(1)中，待测样本的RNA的3’末端加poly(A)通过Poly(A)聚合酶实现。
所述步骤(1)中，待测样本的RNA的3’末端加poly(A)的反应体系包括Poly(A)聚合酶、Poly(A)聚合酶反应Buffer、dATP和RNase inhibitor。
所述步骤(1)中，待测样本的RNA的3’末端加poly(A)的反应条件为：35-39℃(如35-37℃、37-39℃、35℃、37℃或39℃)孵育20-40min(如20-30min、30-40min、20min、30min或40min)。
所述Poly(A)聚合酶优选为E.coli Poly(A)Polymerase。
所述步骤(2)中，进行反转录的引物从5’末端至3’末端可包括DNA片段甲和DNA片段丙，本领域技术人员可以理解DNA片段甲和DNA片段丙的序列长度在不影响其功能的情况下可以任意选择。优选的，DNA片段甲可为15-20bp(如15-18bp、18-20bp、15bp、18bp或20bp)的任意序列。优选的，DNA片段丙从5’末端至3’末端可包括20-30个(如20-25个、25-30个、20个、25个或30个)T和1个V。优选的，DNA片段丙可由20-30个(如20-25个、25-30个、20个、25个或30个)T和1个V组成。优选的，DNA片段丙还可包括1个N，其位于所述V的3’末端。优选的，DNA片段丙从5’末端至3’末端包括20-30个(如20-25个、25-30个、20个、25个或30个)T、1个V和1个N。优选的，DNA片段丙可由20-30个(如20-25个、25-30个、20个、25个或30个)T、1个V和1个N组成。
所述N为A、T、C和G中的任一种，V为A、C和G中的任一种。
所述步骤(2)中，进行反转录的引物(从5’末端至3’末端)可由所述DNA片段甲和所述DNA片段丙组成。
所述步骤(2)中，所述进行反转录的引物还可包括DNA片段乙，DNA片段乙位于DNA片段甲和片段丙的中间，本领域技术人员可以理解DNA片段乙的序列长度在不影响其功能的情况下可以任意选择。优选的，DNA片段乙可为4-16bp(如4-8bp、8-12bp、12-16bp、4bp、8bp、12bp或16bp)的Barcode序列，用于区分不同的待测样本。
所述步骤(2)中，进行反转录的引物从5’末端至3’末端包括所述 DNA片段甲、所述DNA片段乙和所述DNA片段丙。
所述步骤(2)中，进行反转录的引物(从5’末端至3’末端)可由所述DNA片段甲、所述DNA片段乙和所述DNA片段丙组成。
上述任一所述DNA片段甲可以作为通用引物序列。所述通用引物序列可为测序接头序列。
所述步骤(2)中，逆转录酶会在cDNA的3’末端添加3个C，即获得3’末端具有C标记的cDNA。
上述任一所述的方法中，完成步骤(2)后、进行步骤(3)之前，还可包括纯化的步骤。
所述步骤(3)中，末端转移酶处理后的cDNA的3’末端可由8-17个(如8-13个、13-17个、8个、13个或17个)B组成，B为T、C和G中的任一种。
所述步骤(3)中，末端转移酶处理的反应体系可包括末端转移酶、末端转移酶反应Buffer和CoCl2。末端转移酶处理的反应体系还可包括dCTP、dGTP或dTTP。
所述步骤(1)中，末端转移酶处理的反应条件为：35-39℃(如35-37℃、37-39℃、35℃、37℃或39℃)孵育1-2h(如1-1.5h、1.5-2h、1h、1.5h或2h)。
所述步骤(4)中，二链合成优选包括PCR扩增反应。
所述步骤(4)中，进行PCR扩增的引物1从5’末端至3’末端可包括DNA片段a、DNA片段b和1个V；DNA片段a可为所述cDNA 3’末端的反向互补序列；DNA片段b可为GGG；进行PCR扩增的引物2可为所述进行反转录的引物5’末端的一部分。
所述进行PCR扩增的引物1从5’末端至3’末端可由所述DNA片段a、所述DNA片段b和1个V组成。
上述任一所述进行PCR扩增的引物1还可包括1个N，其位于所述V的3’末端。
上述任一所述进行PCR扩增的引物1从5’末端至3’末端包括所述DNA片段a、所述DNA片段b、1个V和1个N。
上述任一所述进行PCR扩增的引物1从5’末端至3’末端可由所述 DNA片段a、所述DNA片段b、1个V和1个N组成。
所述N为A、T、C和G中的任一种，V为A、C和G中的任一种。
所述进行PCR扩增的引物2可为进行反转录的引物或所述DNA片段甲或所述DNA片段甲5’末端的一部分。
上述任一所述V可为A、C和G中的任一种。
上述任一所述N可为A、T、C和G中的任一种。
所述步骤(4)中，进行PCR扩增的引物对由所述引物1和所述引物2组成。
上述任一所述的方法中，完成步骤(4)后、进行步骤(5)之前，还可包括纯化的步骤。
在本发明的实施例中，进行反转录的引物的核苷酸序列具体可为5’-CGACATGGCTACGATCCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’。进行PCR扩增的引物1的核苷酸序列具体可为5’-GGGGGGGGGGGGGGGGGGGGVN-3’，其中N为A、T、C和G中的任一种，V为A、C和G中的任一种。进行PCR扩增的引物2的核苷酸序列具体可为5’-CGACATGGCTACGATCCGACTT-3’。
上述任一所述构建方法还可包括去除rRNA的步骤；去除rRNA可在步骤(1)中3’末端加poly(A)之前进行，也可在步骤(4)获得双链cDNA之后进行。
所述去除rRNA可通过去除rRNA试剂完成。
所述步骤(5)中，获得RNA测序文库还进一步包括如下步骤：
(c1)取cDNA产物或去除rRNA的cDNA产物，片段化；
(c2)末端修复加A；
(c3)连接接头；
(c4)PCR扩增；
(c5)单链环化；
(c6)酶切消化。
所述步骤(5)中，完成步骤(c1)后、进行步骤(c2)之前，还可包括纯化的步骤。
所述步骤(5)中，完成步骤(c3)后、进行步骤(c4)之前，还可 包括纯化的步骤。
所述步骤(5)中，完成步骤(c4)后、进行步骤(c5)之前，还可包括纯化的步骤。
所述步骤(5)中，完成步骤(c4)后、进行步骤(c5)之前，还可包括核酸定量的步骤。完成核酸定量后，还可包括纯化的步骤。
所述步骤(c5)中，进行单链环化的目的可为将单链线性DNA进行环化变成单链环状DNA文库。
所述步骤(c6)中，进行酶切消化的目的可为消化没有环化的单链线性DNA和/或双链线性DNA。
所述步骤(5)中，完成步骤(c6)后，还可包括纯化的步骤。
所述步骤(5)中，完成步骤(c6)后，还可包括核酸定量的步骤。
所述步骤(5)中，建库完成后还可包括测序。所述测序可采用高通量测序仪(如MGISEQ-2000测序仪)进行。
上述任一所述纯化可采用agencourt AMPure XP磁珠实现。纯化后的核酸可溶于TE缓冲液。
上述任一所述的构建方法中，所述待测样本可为细胞、组织或体液等任何生物样本。在本发明的实施例中，所述组织具体可为小鼠脑组织。
本发明还保护上述任一所述的构建方法在分析待测样本RNA中的应用。所述RNA可为mRNA、lncRNA、small RNA、cfRNA和tRNA中的至少一种。所述small RNA为miRNA、siRNA和piRNA中的至少一种。
本发明还保护一种RNA文库构建试剂盒，可包括Poly(A)聚合酶、dATP、末端转移酶、反转录酶、上述任一所述进行反转录的引物、上述任一所述进行PCR扩增的引物1和/或上述任一所述进行PCR扩增的引物2。
所述试剂盒具体可由Poly(A)聚合酶、dATP、末端转移酶、反转录酶、上述任一所述进行反转录的引物、上述任一所述进行PCR扩增的引物1和上述任一所述进行PCR扩增的引物2组成。
上述任一所述试剂盒还可包括上述任一所述去除rRNA试剂和/或建库试剂。
上述任一所述试剂盒具体可由Poly(A)聚合酶、dATP、末端转移酶、反转录酶、上述任一所述进行反转录的引物、上述任一所述进行PCR扩增 的引物1、上述任一所述进行PCR扩增的引物2、去除rRNA试剂和建库试剂组成。
本发明还保护上述任一所述的试剂盒的应用，可为T1)或T2)：
T1)构建待测样本RNA测序文库；
T2)分析待测样本RNA。
上述应用中，所述待测样本可为细胞、组织或体液等任何生物样本。在本发明的实施例中，所述组织具体可为小鼠脑组织。
上述任一所述RNA可为mRNA、lncRNA、small RNA、cfRNA和tRNA中的至少一种。
上述任一所述small RNA可为miRNA、siRNA和piRNA中的至少一种。
本发明先使用具有给RNA加上poly(A)功能的酶给RNA加上poly(A)，再通过带有oligo dT引物和逆转录酶进行逆转录，之后使用末端转移酶给cDNA的3’端加上固定序列作为引物锚定位点的方法，可以同时对多种RNA进行建库，包括带poly(A)的长片段的mRNA和lncRNA、不同poly(A)的长片段的lncRNA以及短片段的不带poly(A)的small RNA。
本发明可以同时对多种RNA(包括mRNA、lncRNA、small RNA、cfRNA和tRNA)进行建库，构建的文库中同时包括mRNA文库、lncRNA文库、small RNA文库、cfRNA文库和tRNA文库，并且过程中不需要特殊的片段回收方法去回收small RNA文库，使得对一份RNA样本只需要建一次库，就可以同时得到多种RNA的信息，不再需要像之前同时研究mRNA和lncRNA以及small RNA时，需要将一份RNA分成几份，单独构建不同RNA的文库，从而提高的RNA建库效率和简便性。本发明具有重要的应用价值。
附图说明
图1为采用传统随机引物逆转录方法构建测序文库的流程示意图。
图2为采用传统Smart-seq建库方法构建测序文库的流程示意图。
图3为采用传统单链接头连接的small RNA建库方法构建测序文库的流程示意图。
图4为基于RNA加polyA尾和末端转移酶的建库方法(即本发明提供的方法)构建测序文库的流程示意图。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中，PCR管的规格均为200μL。离心管的规格均为1.5mL。
下述实施例中涉及的小鼠具体为C57BL/6J。
MGIEasy rRNA去除试剂盒为深圳华大智造科技有限公司的产品，货号为1000005953。
Nuclease Free Water为AMBION公司的产品，货号为AM9932。
实施例1、构建小鼠脑组织RNA测序文库
本发明的发明人经过大量实验，建立了小鼠脑组织的RNA测序文库的构建方法。具体步骤如下：
一、小鼠去除rRNA的RNA的获得
1、取小鼠脑组织，采用RNeasy Mini Kit(Qiagen)提取total RNA，得到小鼠脑组织的total RNA。
2、使用Agilent 2100 Bioanalyzer对小鼠脑组织的total RNA进行质控。
结果表明，小鼠脑组织的total RNA的RIN值≥7。
3、取200ng小鼠脑组织的total RNA，采用MGIEasy rRNA去除试剂盒去除total RNA中的rRNA(具体按照试剂盒的说明书操作)，最后用10μL水溶解，得到小鼠去除rRNA的RNA。
小鼠去除rRNA的RNA中含有带poly(A)的mRNA和不带poly(A)的RNA，不带poly(A)的RNA如lncRNA、tRNA、miRNA、piRNA。
二、3’末端加poly(A)
1、取PCR管，加入2μL 10×E.coli Poly(A)Polymerase Reaction Buffer、1μL E.coli Poly(A)Polymerase、1μL RNase inhibitor(BGI，01E019MM)、2μL dATP(10mM)(Invitrogen，AM8110G)和4μL Nuclease Free Water，混匀。
E.coli Poly(A)Polymerase为NEB公司的产品，货号为M0276S。 10×E.coli Poly(A)Polymerase Reaction Buffer为E.coli Poly(A)Polymerase中的组件。
2、完成步骤1后，取所述PCR管，加入步骤一获得的小鼠去除rRNA的RNA，混匀。
3、完成步骤2后，将所述PCR管置于PCR仪，37℃孵育30min；然后加入3μL浓度为0.5M的EDTA水溶液终止反应，得到小鼠加poly(A)的RNA。
三、逆转录反应
1、取PCR管，加入4μL 5×First Strand Buffer(BGI，01E022MS)、1μL 25mM dNTP(Thermo Scientific，R1121)、1μL RNase Inhibitor(40U/μL)(BGI，01E022MS)、2μL 0.1M DTT(BGI，01E022MS)、1μL Alpha Reverse Transcriptase(200U/μL)(BGI，01E019MM)、1μL RT Primer水溶液(浓度为10μM)和10μL Nuclease Free Water混合，得到混合试剂1。
RT primer的核苷酸序列为：
5’-CGACATGGCTACGATCCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’。
2、完成步骤1后，取完成步骤二中3的PCR管(其中含有小鼠加poly(A)的RNA)，加入混合试剂1，混匀。
3、完成步骤2后，将所述PCR管置于PCR仪，37℃孵育30min，45℃孵育10min，55℃孵育10min，65℃孵育10min。
4、向完成步骤3的反应体系中加入45μL agencourt AMPure XP磁珠(Beackman公司，货号A63881)进行纯化，纯化产物溶于22μL TE缓冲液，回收20μL的逆转录产物。
逆转录酶(即Alpha Reverse Transcriptase)会在逆转录产物的3’末端添加3个C，获得3’末端具有C标记的cDNA。
四、末端转移酶处理
1、取PCR管，加入4μL 10×Terminal Transferase Reaction Buffer、0.5μL Terminal Transferase、0.5μL 10mM dCTP(Invitrogen，18253013)、4μL 2.5mM CoCl 2(NEB，M0315S)和11μL Nuclease Free Water，混匀。
Terminal Transferase为NEB公司的产品，货号为M0315S。 10×Terminal Transferase Reaction Buffer为Terminal Transferase中的组件。
2、完成步骤1后，取所述PCR管，加入步骤三中4回收的逆转录产物，混匀。
3、完成步骤2后，将所述PCR管置于PCR仪，37℃孵育1.5h。
末端转移酶(即Terminal Transferase)会在产物3’末端的C后面加上若干个C。
五、cDNA扩增
1、取PCR管，加入50μL KAPA HiFi Hotstart Ready mix(Kapa Biosystems，KK2602)、2.5μL cDNA PCR Primer-F(浓度为10μM)、2.5μL cDNA PCR Primer-R(浓度为10μM)和5μL Nuclease Free Water，混匀，得到混合试剂2。
cDNA PCR Primer-F的核苷酸序列为5’-GGGGGGGGGGGGGGGGGGGGVN-3’，其中N为A、T、C和G中的任一种，V为A、C和G中的任一种。
cDNA PCR Primer-R的核苷酸序列为5’-CGACATGGCTACGATCCGACTT-3’。
2、完成步骤1后，将混合试剂2加入完成步骤四中3的反应体系中，混匀。
3、完成步骤2后，将所述PCR管置于PCR仪，进行PCR扩增，得到cDNA扩增产物。
反应程序为：95℃2min；95℃10s，60℃20s，72℃3min，15个循环；72℃5min；4℃保持。
六、cDNA扩增产物的纯化
1、将步骤五中3得到的cDNA扩增产物转移至离心管中，再加入150μL agencourt AMPure XP磁珠进行纯化，纯化产物溶于23μL TE缓冲液，回收21μL纯化的cDNA。
2、完成步骤1后，取1μL纯化的cDNA，使用Qubit dsDNA HS Assay kit(Invitrogene)测定浓度。
七、小鼠脑组织RNA测序文库的获得
Fragmentase、10×Fragmentase buffer、ERAT Buffer、ERAT Enzyme Mix、Ligation Buffer、Adapter、DNA Ligase、PCR Primer Mix、Splint Buffer、DNA Rapid Ligase、Digestion Buffer、Digestion Enzyme和Digestion Stop Buffer均为MGIEasy通用DNA文库制备试剂套装中的组件。MGIEasy通用DNA文库制备试剂套装为MGI公司的产品，货号为1000006985。
在冰上配制片段化反应液、末端修复反应液、接头连接反应液、PCR反应混合液、单链环化反应液和酶切消化反应液。
片段化反应液为4μL，由2μL Fragmentase和2μL 10×Fragmentase buffer混合而成。
末端修复反应液为10μL，由7.1μL ERAT Buffer和2.9μL ERAT Enzyme Mix混合而成。
接头连接反应液为30μL，由23.4μL Ligation Buffer、5μL Adapter和1.6μL DNA Ligase混合而成。
PCR反应混合液为30μL，由25μL PCR Enzyme Mix和5μL PCR Primer Mix混合而成。
单链环化反应液为12.1μL，由11.6μL Splint Buffer和0.5μL DNA Rapid Ligase混合而成。
酶切消化反应液为4μL，由1.4μL Digestion Buffer和2.6μL Digestion Enzyme混合而成。
使用MGIEasy通用DNA文库制备试剂套装建库，具体步骤如下：
1、取PCR管，加入16μL纯化的cDNA(含100ng纯化的cDNA)和4μL片段化反应液，混匀。
2、完成步骤1后，将所述PCR管置于PCR仪(热盖设为75℃)，37℃孵育10min；反应结束后加入TE缓冲液至总体积为50μL，混匀后瞬时离心将反应液收集至管底。
进行步骤1和步骤2的目的是将纯化的cDNA进行片段化。
3、完成步骤2后，取所述PCR管，加入75μL agencourt AMPure XP磁珠进行纯化，纯化产物溶于42μL TE缓冲液，回收40μL片段化产物。
4、完成步骤3后，取PCR管，加入10μL末端修复反应液和40μL片段化产物，混匀。
5、完成步骤4后，将所述PCR管置于PCR仪(热盖设为75℃)，37℃孵育30min，65℃孵育15min，4℃保持，得到末端修复产物。
进行步骤4和步骤5的目的是末端修复加“A”。
6、完成步骤5后，取所述PCR管，加入30μL接头连接反应液，混匀。
7、完成步骤6后，将所述PCR管置于PCR仪(热盖设为75℃)，23℃孵育60min，4℃保持。
进行步骤6和步骤7的目的是接头连接。
8、完成步骤7后，取所述PCR管，加入TE缓冲液至总体积为100μL，然后使用100μL agencourt AMPure XP磁珠进行纯化，纯化产物溶于22μL TE缓冲液，回收20μL接头连接产物。
9、完成步骤8后，取PCR管，加入20μL接头连接产物和30μL PCR反应混合液，混匀。
10、完成步骤9后，将所述PCR管置于PCR仪，进行PCR扩增，得到PCR扩增产物。
反应程序为：95℃3min；98℃20s，60℃20s，72℃30s，13个循环；72℃5min；4℃保持。
11、完成步骤10后，取PCR扩增产物，加入75μL agencourt AMPure XP磁珠进行纯化，纯化产物溶于32μL TE缓冲液，回收30μL PCR纯化产物。
12、使用
Figure PCTCN2020079434-appb-000001
dsDNA HS Assay Kit(Invitrogen公司的产品，货号为Q32851)或Quant-iTTM
Figure PCTCN2020079434-appb-000002
dsDNA Assay Kit(Invitrogen公司的产品，货号为P11496)等双链DNA荧光定量试剂盒对步骤11回收的PCR纯化产物进行定量。
结果表明，步骤11回收的PCR纯化产物为350ng。
13、另取一新的PCR管，加入152ng PCR纯化产物，然后加入TE缓冲液补充至总体积为48μL。
14、完成步骤13后，将所述PCR管置于PCR仪，95℃5min，反应结束后，立即将PCR管转移到冰上，静置2min后瞬时离心。
15、完成步骤14后，取所述PCR管，加入12.1μL单链环化反应液，涡旋震荡3次，每次3s，瞬时离心将反应液收集至管底。
16、完成步骤15后，将所述PCR管置于PCR仪，37℃反应30min，反应 结束后，瞬时离心，将PCR管转移到冰上。
进行步骤15和步骤16的目的是将单链线性DNA进行环化变成单链环状DNA文库。
17、完成步骤16后，取所述PCR管，加入4μL酶切消化反应液，涡旋震荡3次，每次3s，瞬时离心将反应液收集至管底。
18、完成步骤17后，将所述PCR管置于PCR仪，37℃反应30min，反应结束后，向PCR管中加入7.5μL Digestion Stop Buffer，涡旋震荡3次，每次3s，瞬时离心将反应液收集至管底，将全部反应液转移到新的离心管中。
进行步骤17和步骤18的目的是消化没有环化的单链线性DNA和双链线性DNA。其中加入Digestion Stop Buffer是终止消化酶作用。
19、完成步骤18后，取所述离心管，加入170μL agencourt AMPure XP磁珠进行纯化，纯化产物溶于22μL TE缓冲液，回收20μL酶切消化产物。
20、完成步骤19后，取酶切消化产物，采用
Figure PCTCN2020079434-appb-000003
ssDNA Assay Kit荧光定量试剂盒进行定量。
结果表明，步骤19回收的酶切消化产物为18ng。
酶切消化产物即为小鼠脑组织RNA测序文库。
八、测序
取小鼠脑组织RNA测序文库，采用MGISEQ-2000高通量测序试剂套装(SE50)(MGI，货号1000012551)进行测序。测序使用MGISEQ-2000测序仪进行。
九、分析
1、测序数据下机后，去掉低质量的reads，进行比对。
根据比对结果，发现小鼠脑组织RNA测序文库中含有miRNA、tRNA、piRNA、mRNA和lncRNA的核苷酸序列。
2、完成步骤1后，将small RNA(如miRNA、piRNA)、mRNA和lncRNA通过TPM(Transcripts Per Million reads，1百万条检测的序列中对应转录本的数目)进行量化。
检测结果见表1。结果表明，采用上述步骤制备的小鼠脑组织RNA测序文库中含有17794个mRNA、526个lncRNA和1105个small RNA。
表1
Figure PCTCN2020079434-appb-000004
需要说明的是，步骤一中rRNA的去除也可以在完成步骤六后、进行步骤七之前进行，此时步骤一为步骤A。rRNA的去除可采用MGIEasy rRNA去除试剂盒进行。
步骤A、小鼠脑组织的total RNA的获得
1、取小鼠脑组织，采用RNeasy Mini Kit(Qiagen)提取total RNA，得到小鼠脑组织的total RNA。
2、使用Agilent 2100 Bioanalyzer对小鼠脑组织的total RNA进行质控。
结果表明，小鼠脑组织的total RNA的RIN值≥7。
小鼠脑组织的total RNA中含有带poly(A)的mRNA和不带poly(A)的RNA，不带poly(A)的RNA如rRNA、lncRNA、tRNA、miRNA、piRNA。
由此可见，本发明提供的方法是一种同时对多种RNA进行文库构建的一种建库技术，核心在于给没有poly(A)的RNA加上poly(A)，使得所有线性RNA都带有poly(A)，再通过带有oligo dT的引物与poly(A)杂交后进行逆转录扩增得到一链cDNA后，使用末端转移酶对cDNA的3’末端加上连续的核苷酸作为cDNA扩增引物来扩增cDNA，随后进行建库。待测样本RNA测序文库的流程示意图见图4。
工业应用
本发明可以同时对多种RNA(包括mRNA、lncRNA、small RNA、cfRNA和tRNA)进行建库，构建的文库中同时包括mRNA文库、lncRNA文库、small RNA文库、cfRNA文库和tRNA文库，并且过程中不需要特殊的片段回收方法去回收small RNA文库，使得对一份RNA样本只需要建一次库，就可以 同时得到多种RNA的信息。本发明提高了RNA建库效率和简便性，具有重要的应用价值。

Claims (14)

  1. RNA测序文库的构建方法，包括如下步骤：
    (1)取待测样本的RNA，3’末端加poly(A)；
    (2)反转录，获得cDNA；
    (3)末端转移酶处理；
    (4)二链合成，获得双链cDNA；
    (5)获得RNA测序文库。
  2. 如权利要求1所述的构建方法，其特征在于：所述步骤(4)中，二链合成包括PCR扩增反应。
  3. 如权利要求1所述的构建方法，其特征在于：所述构建方法还包括去除rRNA的步骤；去除rRNA可在步骤(1)中3’末端加poly(A)之前进行，也可在步骤(4)获得双链cDNA之后进行。
  4. 如权利要求1所述的构建方法，其特征在于：
    所述步骤(2)中，进行反转录的引物从5’末端至3’末端包括DNA片段甲和DNA片段丙；
    DNA片段甲为15-20bp的任意序列；
    DNA片段丙从5’末端至3’末端包括20-30个T和1个V；
    优选的，DNA片段丙由20-30个T和1个V组成或由20-30个T、1个V和1个N组成；
    N为A、T、C和G中的任一种；
    V为A、C和G中的任一种。
  5. 如权利要求4所述的构建方法，其特征在于：
    所述进行反转录的引物还包括DNA片段乙，DNA片段乙位于DNA片段甲和片段丙的中间；DNA片段乙为4-16bp的Barcode序列，用于区分不同的待测样本。
  6. 如权利要求1所述的构建方法，其特征在于：
    所述步骤(3)中，末端转移酶处理后的cDNA的3’末端由8-17个B组成，B为T、C和G中的任一种。
  7. 如权利要求2所述的构建方法，其特征在于：
    所述步骤(4)中，进行PCR扩增的引物1从5’末端至3’末端包括 DNA片段a、DNA片段b和1个V；
    优选的，进行PCR扩增的引物1从5’末端至3’末端包括DNA片段a、DNA片段b、1个V和1个N；
    N为A、T、C和G中的任一种；
    V为A、C和G中的任一种；
    DNA片段a为权利要求6中所述cDNA 3’末端的反向互补序列；
    DNA片段b为GGG；
    进行PCR扩增的引物2为权利要求4或5中进行反转录的引物或所述DNA片段甲或所述DNA片段甲5’末端的一部分。
  8. 如权利要求1所述的构建方法，其特征在于：所述步骤(5)中，获得RNA测序文库进一步包括如下步骤：
    (c1)取cDNA产物或去除rRNA的cDNA产物，片段化；
    (c2)末端修复加A；
    (c3)连接接头；
    (c4)PCR扩增；
    (c5)单链环化；
    (c6)酶切消化。
  9. 权利要求1至8任一所述的构建方法在分析待测样本RNA中的应用；所述RNA为mRNA、lncRNA、small RNA、cfRNA和tRNA中的至少一种；所述small RNA为miRNA、siRNA和piRNA中的至少一种。
  10. 一种RNA文库构建试剂盒，包括Poly(A)聚合酶、dATP、末端转移酶、反转录酶、权利要求4或5中所述进行反转录的引物、权利要求7中所述进行PCR扩增的引物1和/或权利要求7中所述进行PCR扩增的引物2。
  11. 如权利要求10所述的试剂盒，其特征在于：所述试剂盒还可包括去除rRNA的试剂和/或建库试剂。
  12. 权利要求10或11所述的试剂盒的应用，为T1)或T2)：
    T1)构建待测样本RNA测序文库；
    T2)分析待测样本RNA。
  13. 如权利要求12所述的应用，其特征在于：所述待测样本为细胞、 组织或体液等任何生物样本。
  14. 如权利要求12所述的应用，其特征在于：所述RNA为mRNA、lncRNA、small RNA、cfRNA和tRNA中的至少一种；所述small RNA为miRNA、siRNA和piRNA中的至少一种。
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CN114457142A (zh) * 2022-02-25 2022-05-10 福建农林大学 一种高效加尾快速扩增植物dsRNA病毒基因组末端序列的方法及其应用
WO2023202030A1 (zh) * 2022-04-20 2023-10-26 深圳大学 一种小分子rna的高通量测序文库构建方法
CN115976163A (zh) * 2022-09-14 2023-04-18 杭州联川生物技术股份有限公司 一种去除rRNA的探针组合物、建库试剂盒和建库方法
CN116200537A (zh) * 2022-10-14 2023-06-02 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种呼吸道合胞病毒a型全基因组测序方法
CN116200537B (zh) * 2022-10-14 2023-09-19 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种呼吸道合胞病毒a型全基因组测序方法
CN116042770A (zh) * 2022-11-01 2023-05-02 苏州京脉生物科技有限公司 尿液中miRNA文库制备和表达定量的方法及试剂盒
CN116042770B (zh) * 2022-11-01 2023-12-01 苏州京脉生物科技有限公司 尿液中miRNA文库制备和表达定量的方法及试剂盒

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