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WO2025039935A1 - Rna strand specific library construction kit and library construction method - Google Patents

Rna strand specific library construction kit and library construction method Download PDF

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Publication number
WO2025039935A1
WO2025039935A1 PCT/CN2024/111783 CN2024111783W WO2025039935A1 WO 2025039935 A1 WO2025039935 A1 WO 2025039935A1 CN 2024111783 W CN2024111783 W CN 2024111783W WO 2025039935 A1 WO2025039935 A1 WO 2025039935A1
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Prior art keywords
reverse transcriptase
library construction
chain
rna
specific library
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Chinese (zh)
Inventor
刘娜
戴广伟
曹振
商曰朋
刘峰
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Yeasen Biotechnology Shanghai Co Ltd
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Yeasen Biotechnology Shanghai Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • 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

Definitions

  • the present application belongs to the field of biotechnology and relates to an RNA chain-specific library construction kit and a library construction method.
  • the first is to add a certain proportion of actinomycin D during reverse transcription to inhibit the DNA polymerase activity of reverse transcriptase with DNA as the template (negative chain transfer), so as to ensure that in the synthesis of the first-chain cDNA, the reverse transcriptase only has the DNA polymerase activity with RNA as the template, and can only produce the first-chain cDNA without other by-products.
  • the present application is to modify the reverse transcriptase to inactivate its DNA polymerase activity (negative strand transfer) using its DNA as a template, and directly discard actinomycin D, so as to make the kit production safer, the kit more stable, and the user more convenient and safer.
  • the present application provides an RNA chain-specific library construction kit, in which the DNA polymerase activity (negative strand transfer) of the reverse transcriptase used with DNA as a template is reduced or even inactivated, thereby achieving the abandonment of actinomycin D.
  • the reverse transcriptase lacks DNA template-dependent DNA polymerase activity.
  • the kit further comprises a fragmentation reaction component, a second strand cDNA synthesis and end repair plus A component, an adapter ligation component, an adapter ligation component and a library amplification component.
  • the second strand cDNA synthesis and end repair plus A component comprises 0.5-1.5 U/ ⁇ L large Klenow fragment of DNA polymerase I, 0.2-1 U/ ⁇ L RNase H, 0.2-1 U/ ⁇ L T4 DNA polymerase, 0.05-1 U/ ⁇ L Taq DNA polymerase, 0.2-1 U/ ⁇ L T4 polynucleotide kinase, 50-250 mM Tris-HCl at pH 7-9, 20-100 mM MgCl 2 , 120-500 mM NaCl, 0.8-15 mM dNTPs, 0.8-15 mM dUTP and 2-100 mM ATP.
  • the reverse transcriptase of the RNA strand-specific library construction kit is a MMLV reverse transcriptase mutant, and the MMLV reverse transcriptase mutant is selected from any one of the proteins described in a1-a2:
  • a1 Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A or T662A;
  • a2 A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1.
  • the reverse transcriptase of the RNA chain-specific library construction kit is a MMLV reverse transcriptase mutant, whose amino acid sequence is shown in SEQ ID NO: 1.
  • the RNA chain-specific library construction kit may be a plate-type kit.
  • the present application provides a dual-mode transcriptome rapid library construction kit, which comprises the components of the RNA chain-specific library construction kit described in the first aspect.
  • the components of the dual-mode transcriptome rapid library construction kit can be the components disclosed in CN115011669A, except for the reverse transcription
  • the DNA template-dependent DNA polymerase activity of the enzyme is reduced, and actinomycin D is not present in the first-strand cDNA synthesis components.
  • the present application provides a plate-type kit, which includes the components of the RNA chain-specific library construction kit described in the first aspect.
  • the plate kit comprises the following components: the fragmentation reaction component, the first-chain cDNA synthesis component, the second-chain cDNA synthesis and end repair plus A component, the adapter connection component, and the library amplification component; each component is arranged in different reaction wells in the same reaction plate.
  • the present application provides an automated sequencing product, which includes the plate-based kit described in the third aspect.
  • the present application provides a MMLV reverse transcriptase mutant selected from any one of the proteins described in a1-a2:
  • a1 Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A, or T662A;
  • a2 A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1.
  • the present application provides an MMLV reverse transcriptase mutant, which, based on the wild-type MMLV reverse transcriptase, has mutations in 6 amino acid sites: K152E, L333P, T382A, G504S, D524A, and T662A, and the amino acid sequence of the MMLV reverse transcriptase mutant is shown in SEQ ID NO: 1.
  • identity refers to the identity of an amino acid sequence or a nucleotide sequence. Percent sequence identity can be calculated by any method known in the art, for example using the BLOSUM62 matrix, with reference to the method described by Henikoff et al. in PNAS, 89(22): 10915-10919 (1992).
  • K152E refers to the mutation of lysine (K) at position 152 of the amino acid sequence of the wild-type MMLV reverse transcriptase to glutamic acid (E).
  • the MMLV reverse transcriptase mutants provided herein also include amino acid sequences having at least 90% sequence identity with the mutant sequence, or amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.
  • substantially the same or “substantially equivalent” means that under the same detection conditions, the deviation of the test values of the enzyme activity and performance of the MMLV reverse transcriptase mutant does not exceed 20%.
  • the present application provides a gene encoding the MMLV reverse transcriptase mutant described in the fifth aspect.
  • the DNA sequence of the MMLV reverse transcriptase mutant is as shown in SEQ ID NO: 2.
  • the present application provides an expression vector of the MMLV reverse transcriptase mutant described in the fifth aspect.
  • the present application provides a host bacterium for expressing the MMLV reverse transcriptase mutant described in the fifth aspect.
  • the present application provides the use of the MMLV reverse transcriptase mutant described in the fifth aspect in a reverse transcription reaction.
  • the present application provides a method for RNA chain-specific library construction, using DNA template-dependent DNA polymerase A reverse transcriptase having a synthase activity less than 30% of the DNA template-dependent DNA polymerase activity of the wild-type reverse transcriptase and wherein actinomycin D is not used during the synthesis of the first strand cDNA.
  • This example uses Universal Reference RNAs (Agilent, catalog number: 740000) to perform the following RNA strand-specific library construction process analysis.
  • Some of the library construction components used in this application are Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) Components of the library preparation kit.
  • the RNA input amount in this example is 10 ng
  • the reverse transcriptase tested is NEB's Ultra TM II Directional RNA Library Prep Kit for NEB Next First Strand Synthesis Enzyme Mix, Novozymes' VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Cat.
  • the 2nd Reaction Module 2 (dUTP) component of the library construction kit (Cat. No. 12310) is used to synthesize the second-strand cDNA containing dUTP.
  • Hieff from Yisheng Biotechnology Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) 2 ⁇ Super II High-Fidelity Mix components, Hieff RNA 384 CDI Primer for The cDNA adapter-ligated products were amplified using primers RP 501 and RP 701 to RP 707 (Cat. No. 12414) for 16 cycles and then amplified using Hieff DNA Selection Beads magnetic beads (perfect alternative to AMPure XPBeads) (Cat. No. 12601) were used for 0.9x magnetic bead purification, then washed twice with 80% ethanol, and finally eluted with 30 ⁇ L of sterile water. The library was then quantified using Qubit. The library yield results are shown in Table 2. The library peak diagram is shown in Figure 2. Lanes 1-8 represent the agarose electrophoresis results of the libraries obtained from each reaction in Experiment No. 1-8 in Table 2.
  • the sequencing analysis of the mRNA chain-specific library constructed with 10ng of total RNA using different reverse transcriptases revealed that, as shown in Table 3, the quality inspection indicators of the conventional sequencing machine for the libraries constructed using the first-chain cDNA reverse transcribed by different reverse transcriptases were not much different; the proportion of alignment to the genome was basically around 98%, the number of gene detections was around 18,000, the number of transcript detections was over 45,000, and the proportion of exon detections was over 93%; however, the proportion of the antisense chain, an important indicator for chain-specific library construction, varied greatly.
  • the chain-specific performance of the experimental number 1 of the kit 12310 with actinomycin D reached over 96%, and there were differences in the chain specificity of the reverse transcriptases of other manufacturers, which may be related to the DNA template of each enzyme.
  • the activity of DNA polymerase (negative strand transfer) was related to the activity of DNA polymerase (negative strand transfer).
  • the most active one was Invitrogen TM SuperScript TM IV Reverse Transcriptase (Cat. No.: 18090200), with nearly 40% of the first-strand cDNA forming the second-strand cDNA during reverse transcription.
  • Example 1 Based on Example 1, we analyzed that the DNA polymerase activity (negative strand transfer) activity of the reverse transcriptase with DNA as the template can be improved to achieve the DNA polymerase activity with RNA as the template without adding actinomycin D. Therefore, the Yisheng Bio Yisheng ZymeEditor platform is based on the published mutants and literature of various reverse transcriptases to find mutants that inhibit the DNA polymerase activity (negative strand transfer) activity of DNA as the template, and confirm that the wild type of murine leukemia virus reverse transcriptase (MMLV reverse transcriptase) has undergone site-directed mutations at 6 sites: K152E, L333P, T382A, G504S, D524A, T662A, and its amino acid sequence is shown in SEQ ID No: 1.
  • MMLV reverse transcriptase wild type of murine leukemia virus reverse transcriptase
  • the coding DNA of the above-mentioned reverse transcriptase mutant was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and its sequence is shown in SEQ ID No: 2.
  • the coding gene SEQ ID No: 2 of the new reverse transcriptase was expressed and purified by conventional methods to obtain a new reverse transcriptase.
  • DDDP DNA-dependent DNA polymerase activity of MMLV reverse transcriptase mutant
  • the enzymatic activity of the mutant reverse transcriptase of the present application is determined by the pyrophosphate quantitative method, with the wild-type MMLV reverse transcriptase as a reference.
  • the principle of determining the DDDP activity of MMLV reverse transcriptase is to use single-stranded circular DNA as a template. After amplification by MMLV reverse transcriptase, the production of DNA is accompanied by the production of pyrophosphate.
  • the fluorescence value produced is proportional to the pyrophosphate, that is, the measured fluorescence value is proportional to the DDDP activity.
  • the pyrophosphate detection kit was purchased from AAT Bioquest.
  • the specific implementation method for testing the DDDP activity of MMLV reverse transcriptase is as follows:
  • Pyrophosphate detection Add 48 ⁇ L DEPC water, 2 ⁇ L mixed enzyme reaction solution, and 50 ⁇ L pyrophosphate working solution to a 96-well ELISA plate. After shaking and mixing with an ELISA reader, incubate at 25°C for 20 minutes, measure the fluorescence value with an ELISA reader, and calculate the activity of the mutant reverse transcriptase based on the fluorescence value.
  • the DDDP activity results of the new reverse transcriptase are shown in Table 5.
  • This example uses Universal Reference RNAs (Agilent, catalog number: 740000) to perform the following RNA strand-specific library construction process analysis.
  • Some of the library construction components used are Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) library construction kit components.
  • the RNA input amount in this example is 1 ⁇ g and 10 ng.
  • the specific library construction process refers to Experimental Groups 1 and 5 of Example 1.
  • the amount of reverse transcriptase used in each reaction of the 12310 kit and the new reverse transcriptase is 200 U.
  • the experimental groups No. 1, 2, 5, and 6 in Table 6 were treated with Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310); Experiment No. 3, 4 & 7, 8, except that the 1st cDNA synthesis reaction buffer (250mM Tris-HCl pH 8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs) does not contain actinomycin D, the synthesis reaction buffer components are the same as those of Experiment No. 1, 2 & 5, 6.
  • the reverse transcriptase used was a new reverse transcriptase and Qiagen's RNAse Inhibitor (Cat. No. Y9240L) for the synthesis of the first-strand cDNA.
  • the library construction yield and sequencing data analysis of the new reverse transcriptase found that the library construction yield was equivalent and the library peak shape was basically consistent as shown in Figure 3.
  • Lanes 1-8 represent the agarose electrophoresis results of the libraries obtained from each reaction in experimental number 1-8 in Table 6.
  • the off-machine quality control of the sequencing data was qualified and basically consistent, but at 10ng input, the antisense strand ratio of the chain-specific library construction quality control indicator was 2-3% higher than that of No. 5 and No. 6 using the 12310 kit.
  • the use of the new reverse transcriptase can indeed achieve a consistent antisense strand ratio of 10ng-1 ⁇ g without using actinomycetes D, which can reach more than 98%.
  • Component A fragmentation reaction component, using the fragmentation reaction component in the 12310 kit;
  • Component B first-strand cDNA synthesis component, specifically 5-25U/ ⁇ L reverse transcriptase, 1-2U/ ⁇ L RNase inhibitor, 150-250mM Tris-HCl at pH 7-9, 200-400mM KCl, 10-20mM DTT, 2.5-15mM dNTPs;
  • Component C Synthesis of second strand cDNA and end repair plus component A, specifically 0.5-1.5U/ ⁇ L large Klenow fragment of DNA polymerase I, 0.2-1U/ ⁇ L RNase H, 0.2-1U/ ⁇ L T4 DNA polymerase, 0.05-1U/ ⁇ L Taq DNA polymerase, 0.2-1U/ ⁇ L T4 polynucleotide kinase, 5-250mM Tris-HCl at pH 7-9, 20-100mM MgCl 2 , 120-500mM NaCl, 0.8-15mM dNTPs, 0.8-15mM dUTP, 2-100mM ATP;
  • Component D adapter ligation component, specifically 50-500 U/ ⁇ L T4 DNA ligase, 50-350 mM Tris-HCl at pH 7-9, 10-100 mM MgCl 2 , 2-10 mM ATP, and 3-10% v/v PEG8000.
  • Component E Library amplification component, using 2 ⁇ Super II High-Fidelity Mix Component.
  • Figure 1 is a schematic diagram of a plate-type RNA strand-specific library construction kit for 96T reactions.
  • the plate-type RNA strand-specific library construction kit is packaged and stored as shown in Figure 1.
  • Components A to E meet the number of 96 reaction samples respectively; specifically, A1-H1 is component A of 12T; and the same applies to other components.
  • FIG5 shows that other reaction components required for automated sequencing are further added to the library construction kit, such as adding a universal adapter or universal amplification primer to column 6.
  • Hieff RNA 384 CDI Primer for (Item No.: 12414)
  • PE Adapter the 7th, 8th, and 9th columns are added with Hieff DNA Selection Beads (a perfect alternative to AMPure XPBeads) (Cat. No. 12601), the number of columns of sorting beads can be increased according to demand, not limited to 3 columns.
  • This layout can be perfectly matched with automation, no need to transfer plates, and can save customers' layout and time, and can also be directly used on different automation platforms.
  • the specific integrated reaction plate can select common reaction plates with different arrangements, such as 8-row PCR reaction plates and 96-well reaction plates. When the number of samples is large and the pre-mixed amount of each component is large, a deep-well plate can be selected to meet the reaction requirements.
  • Example 5 Comparative test of plate-type RNA strand-specific library construction kit and 12310 kit
  • RNA input amount in this example is 1 ⁇ g, 10 ng.
  • the fragmentation reaction buffer of the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) was used for fragmentation at 94°C for 7 min.
  • the fragmentation reaction buffer of the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) was used for fragmentation at 94°C for 7 min.
  • the first-strand cDNA synthesis component 2.0 (component B) of the plate-type strand-specific library construction kit contains: 25U/ ⁇ L novel reverse transcriptase, 2U/ ⁇ L RNase inhibitor, 250mM Tris-HCl, pH 8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs. Configure the first-strand cDNA synthesis reaction system according to Table 7.
  • the reaction conditions were: 25°C for 10 min, 42°C for 15 min, 70°C for 15 min, and storage at 4°C.
  • the first-strand cDNA synthesis was completed using two kits: 12310 and Plate-type Strand-Specific Library Construction Kit.
  • the second strand cDNA synthesis component 2.0 (component C) of the plate-type strand-specific library construction kit includes: 1.5 U/ ⁇ L large Klenow fragment of DNA polymerase I, 0.25 U/ ⁇ L RNase H, 0.2 U/ ⁇ L T4 DNA polymerase, 1 U/ ⁇ L Taq DNA polymerase, 1 U/ ⁇ L T4 polynucleotide kinase, 100 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 , 100 mM NaCl, 0.8 mM dNTPs, 2 mM ATP, 0.8 mM dUTP.
  • the second strand cDNA synthesis reaction system was configured according to Table 8.
  • the reaction conditions were: 16°C for 30 min, 72°C for 15 min, and stored at 4°C.
  • Adapter Ligation Component 2.0 contains: 500 U/ ⁇ L Novel T4 DNA ligase, 350 mM Tris-HCl at pH 8.0, 16 mM MgCl 2 , 2.8 mM ATP, 10% PEG8000 (v/v). According to Table 9, the cDNA adapter ligation system was prepared.
  • Hieff RNA 384CDIPrimer for The cDNA adapter ligation products were amplified using RP 501 and RP 701 to RP 707 amplification primers (Cat. No. 12414).
  • the total input RNA was 1 ⁇ g and the number of amplification cycles was 10 cycles.
  • the total input RNA was 10 ng and the number of amplification cycles was 16 cycles.
  • the plate-type chain-specific library construction kit was about 1% higher than 12310; the proportion of exons in the plate-type chain-specific library construction kit was slightly higher than 12310 at different input amounts; the key quality inspection indicator of chain-specific library construction, the proportion of antisense chain, was equivalent at 1 ⁇ g, both reaching more than 98.5%, and at 10ng, the plate-type chain-specific library construction kit was higher than 12310, and the plate-type chain-specific library construction kit was higher than 12310, and the plate-type chain-specific library construction kit was higher than 12310.
  • the library kit has consistent strand specificity at different input amounts. According to the above case, the plate-type strand-specific library construction kit can achieve a library construction output and sequencing data index of 12310 in terms of library construction performance
  • RNAs (Agilent, catalog number: 740000) and total RNA from 293 cell lines were used to perform automated testing of plate-based RNA strand-specific library construction as shown below.
  • the plate-based RNA strand-specific library construction kit is packaged and stored as shown in Figure 1.
  • the automation instrument uses the MGISP-960 high-throughput automated sample preparation system. Automated library construction was performed according to Yishen's automated library construction process operation instructions. Because the layout of the plate-based reagents in this embodiment only occupies columns 1-5 (corresponding to components A to E in Figure 5, respectively), and columns 6-12 are empty wells, we can add universal adapters or universal amplification primers to column 6 according to Figure 5 when preparing reagents before automation.
  • Hieff RNA 384 CDI Primer for PE Adapter (Cat. No.: 12414) (corresponding to Primer Mix in Figure 5), the 7th, 8th, and 9th columns are added with Hieff DNA Selection Beads (a perfect replacement for AMPure XPBeads) (Cat. No. 12601) (corresponding to the three rows of DNA purification beads in Figure 5).
  • This layout can be perfectly matched with automation, without the need for plate transfer, and can save customers layout and time, and can also be directly used on different automation platforms.
  • the plate-type chain-specific library construction kit of this embodiment was subjected to two rows of automated testing, A1-A12 (mRNA capture step 1 ⁇ g Universal Reference RNAs were used to construct mRNA libraries for samples A1-A12 (12 numbers in the sample labeling number in the mRNA capture step), and 1 ⁇ g 293 total RNA was used to construct mRNA libraries for samples B1-B12 (12 numbers in the sample labeling number in the mRNA capture step).
  • Samples A1-A12 were added to row A of the plate-type strand-specific library construction kit, and the reactions were carried out in sequence according to the library construction reaction steps; samples B1-B12 were added to row B of the plate-type strand-specific library construction kit, and the reactions were carried out in sequence according to the library construction reaction steps.
  • Table 12 shows the library yield and sequencing analysis of Universal Reference RNAs A1-A12
  • Table 13 shows the library yield and sequencing data analysis of 293 RNAs B1-B12.
  • Example 7 Stability test of plate-type RNA strand-specific library construction kit
  • RNA chain-specific library construction kit is packaged in the form shown in Figure 1. After being placed at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, and 25°C for 2 days, 4 days, 6 days, 10 days, and 14 days, 1 ⁇ g of library construction and sequencing analysis were performed according to the plate-type RNA library construction process of Example 4.

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Abstract

An RNA strand specific library construction kit, comprising a first strand cDNA synthesis component, wherein the first strand cDNA synthesis component comprises at least reverse transcriptase, the activity of DNA template-dependent DNA polymerase of the reverse transcriptase is 30% or less of the activity of DNA template-dependent DNA polymerase of wild type reverse transcriptase, and the first strand cDNA synthesis component does not contain actinomycin D. The amino acid sequence of an MMLV reverse transcriptase mutant is as shown in SEQ ID NO: 1. The activity of DNA template-dependent DNA polymerase of the MMLV reverse transcriptase mutant is inactivated, and in the synthesis of a first strand cDNA, the RNA strand specific library construction kit prepared by the MMLV reverse transcriptase mutant does not need to additionally add the actinomycin D, such that the stability, safety and convenience of the kit can be improved.

Description

RNA链特异性建库试剂盒及建库方法RNA chain specific library construction kit and library construction method 技术领域Technical Field

本申请属于生物技术领域，涉及RNA链特异性建库试剂盒及建库方法。The present application belongs to the field of biotechnology and relates to an RNA chain-specific library construction kit and a library construction method.

背景技术Background Art

随着第二代测序技术的快速发展，越来越多的科学家在利用第二代测序技术来解析各种生物学问题。第二代测序可以进行全基因组层面的重测序，分析基因组层面的差异；也可以进行转录组层面的测序，解析细胞或者特定的组织在某个发育阶段的基因表达谱。转录组测序又分为链特异性建库和非链特异性建库。链特异性建库又称ssRNA-seq library，可以区分转录本是来自于正义链还是反义链，这样可以更好的帮助科研者区分转录本，确认基因表达的信息，更好的解释科学问题。With the rapid development of second-generation sequencing technology, more and more scientists are using it to solve various biological problems. Second-generation sequencing can perform whole-genome resequencing to analyze differences at the genome level; it can also perform transcriptome sequencing to analyze the gene expression profile of cells or specific tissues at a certain developmental stage. Transcriptome sequencing is divided into strand-specific library construction and non-strand-specific library construction. Strand-specific library construction, also known as ssRNA-seq library, can distinguish whether the transcript comes from the sense strand or the antisense strand, which can better help researchers distinguish transcripts, confirm gene expression information, and better explain scientific problems.

目前可以实现链特异性的建库的策略有三点必须同时满足才能完成链特建库。第一个是在逆转录的过程中，添加一定比例的放线菌素D，抑制逆转录酶的DNA为模板的DNA聚合酶活性(负链转移)，保证在第一链cDNA的合成过程中，逆转录酶只有以RNA为模板的DNA聚合酶活性，只能产生第一链cDNA，无其他副产物产生，但是放线菌素D是有很多缺点的，比如对光非常敏感，容易出现见光降解，需要避光保存，造成试剂不稳定性、试剂使用简便性降低；比如有毒，虽然使用浓度较低，但是对生产等也存在一定风险；比如会逐渐吸附在塑料和玻璃的表面，这会造成操作时间过长，放线菌素D失效明显，因此有些产品甚至建议使用者现用现配。第二个是在第二链cDNA合成中添加dUTP，使合成的第二链cDNA中是含有dUTP的DNA链。第三个是利用UDGase，在PCR前将含有dUTP的第二链cDNA消化掉，保证只有第一链cDNA进入文库扩增。At present, there are three points that must be met simultaneously in the strategy of chain-specific library construction to complete chain-specific library construction. The first is to add a certain proportion of actinomycin D during reverse transcription to inhibit the DNA polymerase activity of reverse transcriptase with DNA as the template (negative chain transfer), so as to ensure that in the synthesis of the first-chain cDNA, the reverse transcriptase only has the DNA polymerase activity with RNA as the template, and can only produce the first-chain cDNA without other by-products. However, actinomycin D has many disadvantages, such as being very sensitive to light, prone to photodegradation, and needing to be stored away from light, resulting in reagent instability and reduced ease of use; for example, it is toxic, and although the concentration used is low, there are certain risks to production, etc.; for example, it will gradually adsorb on the surface of plastic and glass, which will cause the operation time to be too long, and actinomycin D will be obviously ineffective, so some products even recommend that users prepare it before use. The second is to add dUTP to the synthesis of the second-chain cDNA, so that the synthesized second-chain cDNA is a DNA chain containing dUTP. The third is to use UDGase to digest the second-strand cDNA containing dUTP before PCR to ensure that only the first-strand cDNA enters the library amplification.

因此，对于增加链特异性建库试剂盒的稳定性、安全性、方便性，亟需对实现链特异性的第一点进行改进，本申请就是通过对逆转录酶进行改造，使其失活其DNA为模板的DNA聚合酶活性(负链转移)，将放线菌素D直接舍弃，让试剂盒生产更安全、使试剂盒更稳定，让使用者更方便、更安全。Therefore, in order to increase the stability, safety and convenience of the chain-specific library construction kit, it is urgent to improve the first point of achieving chain specificity. The present application is to modify the reverse transcriptase to inactivate its DNA polymerase activity (negative strand transfer) using its DNA as a template, and directly discard actinomycin D, so as to make the kit production safer, the kit more stable, and the user more convenient and safer.

发明内容Summary of the invention

本申请提供了一种RNA链特异性建库试剂盒，其中所使用的逆转录酶的DNA为模板的DNA聚合酶活性(负链转移)降低，甚至失活，实现了舍弃放线菌素D。The present application provides an RNA chain-specific library construction kit, in which the DNA polymerase activity (negative strand transfer) of the reverse transcriptase used with DNA as a template is reduced or even inactivated, thereby achieving the abandonment of actinomycin D.

第一方面，本申请提供了一种RNA链特异性建库试剂盒，至少包含逆转录酶和第一链cDNA合成组分，所述逆转录酶依赖DNA模板的DNA聚合酶活性在野生型逆转录酶依赖DNA模板的DNA聚合酶活性的30％以下，所述第一链cDNA合成组分中不含放线菌素D。降低或缺失逆转录酶的依赖DNA模板的DNA聚合酶活性可以通过酶突变、功能域除去、抗体封闭等方式实现。In the first aspect, the present application provides an RNA strand-specific library construction kit, comprising at least a reverse transcriptase and a first strand cDNA synthesis component, wherein the reverse transcriptase has a DNA template-dependent DNA polymerase activity that is less than 30% of the DNA template-dependent DNA polymerase activity of a wild-type reverse transcriptase, and the first strand cDNA synthesis component does not contain actinomycin D. Reducing or deleting the DNA template-dependent DNA polymerase activity of the reverse transcriptase can be achieved by enzyme mutation, functional domain removal, antibody blocking, and the like.

优选地，所述逆转录酶缺失依赖DNA模板的DNA聚合酶活性。 Preferably, the reverse transcriptase lacks DNA template-dependent DNA polymerase activity.

优选地，所述试剂盒还包含第一链cDNA合成组分，所述第一链cDNA合成组分包括5-25U/μL逆转录酶、1-2U/μL RNase抑制剂、150-250mM pH＝7-9的Tris-HCl、200-400mM KCl、10-20mM DTT和2.5-15mM dNTPs。Preferably, the kit also contains first-strand cDNA synthesis components, which include 5-25U/μL reverse transcriptase, 1-2U/μL RNase inhibitor, 150-250mM Tris-HCl with pH=7-9, 200-400mM KCl, 10-20mM DTT and 2.5-15mM dNTPs.

优选地，所述第一链cDNA合成组分包括25U/μL逆转录酶、2U/μL RNase抑制剂、250mM pH＝8.3的Tris-HCl、300mM KCl、20mM DTT和2.5mM dNTPs。Preferably, the first-strand cDNA synthesis components include 25U/μL reverse transcriptase, 2U/μL RNase inhibitor, 250mM Tris-HCl (pH=8.3), 300mM KCl, 20mM DTT and 2.5mM dNTPs.

优选地，所述试剂盒还包括片段化反应组分、第二链cDNA的合成及末端修复加A组分、接头连接组分、接头连接组分和文库扩增组分。Preferably, the kit further comprises a fragmentation reaction component, a second strand cDNA synthesis and end repair plus A component, an adapter ligation component, an adapter ligation component and a library amplification component.

优选地，所述第二链cDNA的合成及末端修复加A组分包括0.5-1.5U/μL DNA聚合酶I的大Klenow片段、0.2-1U/μL核糖核酸酶H、0.2-1U/μL T4 DNA聚合酶、0.05-1U/μL Taq DNA聚合酶、0.2-1U/μL T4多聚核苷酸激酶、50-250mM pH＝7-9的Tris-HCl、20-100mM MgCl2、120-500mM NaCl、0.8-15mM dNTPs、0.8-15mM dUTP和2-100mM ATP。Preferably, the second strand cDNA synthesis and end repair plus A component comprises 0.5-1.5 U/μL large Klenow fragment of DNA polymerase I, 0.2-1 U/μL RNase H, 0.2-1 U/μL T4 DNA polymerase, 0.05-1 U/μL Taq DNA polymerase, 0.2-1 U/μL T4 polynucleotide kinase, 50-250 mM Tris-HCl at pH 7-9, 20-100 mM MgCl 2 , 120-500 mM NaCl, 0.8-15 mM dNTPs, 0.8-15 mM dUTP and 2-100 mM ATP.

优选地，所述第二链cDNA的合成及末端修复加A组分包括1.5U/μL DNA聚合酶I的大Klenow片段、0.25U/μL核糖核酸酶H、0.2U/μL T4 DNA聚合酶、1U/μL Taq DNA聚合酶、1U/μL T4多聚核苷酸激酶、100mM pH＝8.0的Tris-HCl、20mM MgCl2、100mM NaCl、0.8mM dNTPs、2mM ATP和0.8mM dUTP。Preferably, the second strand cDNA synthesis and end repair plus A component includes 1.5 U/μL large Klenow fragment of DNA polymerase I, 0.25 U/μL RNase H, 0.2 U/μL T4 DNA polymerase, 1 U/μL Taq DNA polymerase, 1 U/μL T4 polynucleotide kinase, 100 mM Tris-HCl, pH=8.0, 20 mM MgCl 2 , 100 mM NaCl, 0.8 mM dNTPs, 2 mM ATP and 0.8 mM dUTP.

优选地，所述接头连接组分包括50-500U/μL的T4 DNA连接酶、50-350mM pH＝7-9的Tris-HCl、10-100mM MgCl2、2-10mM ATP和3-10％v/v的PEG8000。Preferably, the linker ligation components include 50-500 U/μL T4 DNA ligase, 50-350 mM Tris-HCl at pH=7-9, 10-100 mM MgCl 2 , 2-10 mM ATP and 3-10% v/v PEG8000.

优选地，所述接头连接组分包括500U/μL的Novel T4DNA连接酶，350mM pH＝8.0的Tris-HCl、16mM MgCl2、2.8mM ATP和10％v/v的PEG8000。Preferably, the adapter ligation component includes 500 U/μL of Novel T4 DNA ligase, 350 mM Tris-HCl, pH=8.0, 16 mM MgCl 2 , 2.8 mM ATP, and 10% v/v PEG8000.

优选地，RNA链特异性建库试剂盒的逆转录酶为MMLV逆转录酶突变体，所述MMLV逆转录酶突变体选自a1-a2中任一所述的蛋白质：Preferably, the reverse transcriptase of the RNA strand-specific library construction kit is a MMLV reverse transcriptase mutant, and the MMLV reverse transcriptase mutant is selected from any one of the proteins described in a1-a2:

a1：其在野生型MMLV逆转录酶的基础上，发生如下位点中的一个或多个位点的突变：K152E、L333P、T382A、G504S、D524A或T662A；a1: Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A or T662A;

a2：与a1的蛋白质具有至少90％的序列同一性，且酶活性及性能与a1的蛋白质基本相当的蛋白质。a2: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1.

优选地，RNA链特异性建库试剂盒的逆转录酶为MMLV逆转录酶突变体，其氨基酸序列如SEQ ID NO：1所示。Preferably, the reverse transcriptase of the RNA chain-specific library construction kit is a MMLV reverse transcriptase mutant, whose amino acid sequence is shown in SEQ ID NO: 1.

所述RNA链特异性建库试剂盒可以采用板式试剂盒。The RNA chain-specific library construction kit may be a plate-type kit.

第二方面，本申请提供了一种双模式转录组快速建库试剂盒，其包含第一方面所述的RNA链特异性建库试剂盒中的各组分。In a second aspect, the present application provides a dual-mode transcriptome rapid library construction kit, which comprises the components of the RNA chain-specific library construction kit described in the first aspect.

双模式转录组快速建库试剂盒的组分可以采用CN115011669A公开的组分，除了逆转录 酶的依赖DNA模板的DNA聚合酶活性降低，以及第一链cDNA合成组分中不含放线菌素D。The components of the dual-mode transcriptome rapid library construction kit can be the components disclosed in CN115011669A, except for the reverse transcription The DNA template-dependent DNA polymerase activity of the enzyme is reduced, and actinomycin D is not present in the first-strand cDNA synthesis components.

第三方面，本申请提供了一种板式试剂盒，其包括第一方面所述的RNA链特异性建库试剂盒中的各组分。In a third aspect, the present application provides a plate-type kit, which includes the components of the RNA chain-specific library construction kit described in the first aspect.

优选地，所述板式试剂盒包括如下组分：所述片段化反应组分、所述第一链cDNA合成组分、第二链cDNA的合成及末端修复加A组分、所述接头连接组分、所述文库扩增组分；各组分分别排布在同一反应板中的不同反应孔中。Preferably, the plate kit comprises the following components: the fragmentation reaction component, the first-chain cDNA synthesis component, the second-chain cDNA synthesis and end repair plus A component, the adapter connection component, and the library amplification component; each component is arranged in different reaction wells in the same reaction plate.

第四方面，本申请提供了一种自动化测序产品，其包括第三方面所述的板式试剂盒。In a fourth aspect, the present application provides an automated sequencing product, which includes the plate-based kit described in the third aspect.

第五方面，本申请提供了一种MMLV逆转录酶突变体，其选自a1-a2中任一所述的蛋白质：In a fifth aspect, the present application provides a MMLV reverse transcriptase mutant selected from any one of the proteins described in a1-a2:

a1：其在野生型MMLV逆转录酶的基础上，发生如下位点中的一个或多个位点的突变：K152E、L333P、T382A、G504S、D524A、或T662A；a1: Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A, or T662A;

a2：与a1的蛋白质具有至少90％的序列同一性，且酶活性及性能与a1的蛋白质基本相当的蛋白质。a2: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1.

优选地，本申请提供的一种MMLV逆转录酶突变体，在野生型MMLV逆转录酶的基础上，所述MMLV逆转录酶突变体具有6个氨基酸位点的突变：K152E、L333P、T382A、G504S、D524A、T662A，所述MMLV逆转录酶突变体的氨基酸序列如SEQ ID NO：1所示。Preferably, the present application provides an MMLV reverse transcriptase mutant, which, based on the wild-type MMLV reverse transcriptase, has mutations in 6 amino acid sites: K152E, L333P, T382A, G504S, D524A, and T662A, and the amino acid sequence of the MMLV reverse transcriptase mutant is shown in SEQ ID NO: 1.

本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可以通过本领域已知的任何方法计算百分比序列同一性，例如使用BLOSUM62 matrix，参照Henikoff等人在PNAS,89(22):10915-10919(1992)中描述的方法。Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. Percent sequence identity can be calculated by any method known in the art, for example using the BLOSUM62 matrix, with reference to the method described by Henikoff et al. in PNAS, 89(22): 10915-10919 (1992).

本申请对逆转录酶突变体的描述是本领域人员认同的描述，以其中的MMLV逆转录酶突变体的序列示例，K152E，指的是野生型MMLV逆转录酶氨基酸序列的第152位的赖氨酸(K)突变为了谷氨酸(E)。The description of reverse transcriptase mutants in this application is a description recognized by those skilled in the art. The sequence of the MMLV reverse transcriptase mutant is exemplified as K152E, which refers to the mutation of lysine (K) at position 152 of the amino acid sequence of the wild-type MMLV reverse transcriptase to glutamic acid (E).

本申请提供的MMLV逆转录酶突变体还包括与突变序列具有至少90％序列同一性的氨基酸序列，或至少95％、96％、97％、98％、99％、99.5％、99.8％序列同一性的氨基酸序列。The MMLV reverse transcriptase mutants provided herein also include amino acid sequences having at least 90% sequence identity with the mutant sequence, or amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.

本文中，“基本相同”或“基本相当”是指在相同的检测条件下，MMLV逆转录酶突变体的酶活性及性能的测试值的偏差不超过20％。Herein, "substantially the same" or "substantially equivalent" means that under the same detection conditions, the deviation of the test values of the enzyme activity and performance of the MMLV reverse transcriptase mutant does not exceed 20%.

第六方面，本申请提供了第五方面所述的MMLV逆转录酶突变体的编码基因。In a sixth aspect, the present application provides a gene encoding the MMLV reverse transcriptase mutant described in the fifth aspect.

优选地，所述MMLV逆转录酶突变体的DNA序列如SEQ ID NO：2所示。Preferably, the DNA sequence of the MMLV reverse transcriptase mutant is as shown in SEQ ID NO: 2.

第七方面，本申请提供了第五方面所述的MMLV逆转录酶突变体的表达载体。In a seventh aspect, the present application provides an expression vector of the MMLV reverse transcriptase mutant described in the fifth aspect.

第八方面，本申请提供了表达第五方面所述的MMLV逆转录酶突变体的宿主菌。In an eighth aspect, the present application provides a host bacterium for expressing the MMLV reverse transcriptase mutant described in the fifth aspect.

第九方面，本申请提供了第五方面所述的MMLV逆转录酶突变体在逆转录反应中的应用。In a ninth aspect, the present application provides the use of the MMLV reverse transcriptase mutant described in the fifth aspect in a reverse transcription reaction.

第十方面，本申请提供了一种RNA链特异性建库方法，采用依赖DNA模板的DNA聚 合酶活性在野生型逆转录酶依赖DNA模板的DNA聚合酶活性的30％以下的逆转录酶，且合成第一链cDNA时不使用放线菌素D。In a tenth aspect, the present application provides a method for RNA chain-specific library construction, using DNA template-dependent DNA polymerase A reverse transcriptase having a synthase activity less than 30% of the DNA template-dependent DNA polymerase activity of the wild-type reverse transcriptase and wherein actinomycin D is not used during the synthesis of the first strand cDNA.

优选地，所述RNA链特异性建库方法步骤包括：Preferably, the RNA chain-specific library construction method comprises the following steps:

(1)将mRNA进行片段化处理；(1) fragmenting mRNA;

(2)以片段化的mRNA为模板，在逆转录酶的作用下合成第一链cDNA；(2) Using the fragmented mRNA as a template, the first-strand cDNA is synthesized under the action of reverse transcriptase;

(3)合成第二链cDNA；(3) Synthesizing the second-strand cDNA;

(4)cDNA连接接头，纯化连接产物；以及(4) ligating the cDNA to an adapter and purifying the ligation product; and

(5)对cDNA与接头的连接产物进行文库扩增，获得RNA链特异性文库。(5) Amplify the library of the ligation products of cDNA and adapter to obtain an RNA chain-specific library.

优选地，所述逆转录酶为上述的MMLV逆转录酶突变体。RNA链特异性建库试剂盒还可以包括片段化反应组分及文库扩增组分，所述片段化反应组分及文库扩增组分可以采用翌圣生物的Hieff Ultima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒中相应的片段化反应组分及文库扩增组分。Preferably, the reverse transcriptase is the MMLV reverse transcriptase mutant described above. The RNA strand-specific library construction kit may also include a fragmentation reaction component and a library amplification component, and the fragmentation reaction component and the library amplification component may be Hieff The corresponding fragmentation reaction components and library amplification components in the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) library construction kit.

相对于现有技术，本申请具有如下有益效果：Compared with the prior art, this application has the following beneficial effects:

本申请的MMLV逆转录酶突变体，其DNA为模板的DNA聚合酶活性(负链转移)失活。用其制备的RNA链特异性建库试剂盒，在合成第一链cDNA时不需要再额外添加放线菌素D，可以显著的增加试剂盒的稳定性、安全性、方便性。The MMLV reverse transcriptase mutant of the present application has its DNA polymerase activity (negative strand transfer) inactivated using DNA as a template. The RNA strand-specific library construction kit prepared using the mutant does not require additional addition of actinomycin D when synthesizing the first strand cDNA, which can significantly increase the stability, safety and convenience of the kit.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本申请的RNA链特异性建库试剂盒板式形式。FIG1 is a plate-type diagram of the RNA strand-specific library construction kit of the present application.

图2为本申请的不同逆转录酶的链特建库文库峰图分析。FIG. 2 is a peak diagram analysis of the chain-specific libraries of different reverse transcriptases of the present application.

图3为本申请的新型的逆转录酶在RNA链特异性建库中的文库峰图分析。FIG3 is a peak diagram analysis of the library of the novel reverse transcriptase of the present application in RNA chain-specific library construction.

图4为本申请的板式链特异性建库试剂盒和12310建库试剂盒的建库文库峰图分析。FIG. 4 is a peak diagram analysis of the library construction of the plate-type chain-specific library construction kit and the 12310 library construction kit of the present application.

图5为本申请的一种板式RNA链特异性建库试剂盒的自动化装液形式。FIG5 is an automated liquid filling format of a plate-type RNA strand-specific library construction kit of the present application.

图6为本申请的板式链特异性建库试剂盒自动化建库A1-A12文库峰图分析。FIG6 is a peak diagram analysis of the A1-A12 libraries constructed automatically by the plate-type chain-specific library construction kit of the present application.

图7为本申请的板式链特异性建库试剂盒自动化建库B1-B12文库峰图分析。FIG. 7 is a peak diagram analysis of the B1-B12 libraries constructed automatically by the plate-type chain-specific library construction kit of the present application.

图8为板式RNA链特异性建库试剂盒的稳定性建库文库峰图分析。FIG8 is a peak analysis of the stability library construction of the plate-type RNA strand-specific library construction kit.

具体实施方式DETAILED DESCRIPTION

下面结合附图对本申请的具体实施方式做进一步说明，但实施例的描述不对本申请的保护范围产生任何限制。The specific implementation modes of the present application are further described below in conjunction with the accompanying drawings, but the description of the embodiments does not impose any limitation on the protection scope of the present application.

除非另有定义，本申请所使用的所有的技术术语和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同，在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by technicians in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

下列实施例中所用的物质或仪器，如果未进行特殊说明的话，均可以从常规的商用渠道获取。Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

实施例1：不同逆转录酶的DNA对链特异性建库的影响Example 1: Effects of DNA from different reverse transcriptases on strand-specific library construction

本实施例是利用Universal Reference RNAs(安捷伦，货号：740000)进行如下所示的RNA链特建库流程分析。本申请使用的部分建库组分是翌圣生物的HieffUltima Dual-mode  RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的组分。本实施例的RNA投入量为10ng，测试的逆转录酶有NEB的UltraTMII Directional RNA Library Prep Kit for的NEB Next First Strand Synthesis Enzyme Mix、诺唯赞的VAHTS Universal V8RNA-seq Library Prep Kit for Illumina(货号：NR605-01)1st Strand Enzyme Mix 3、InvitrogenTM的SuperScriptTMII反转录酶(货号：18064022)、SuperScriptTMIII逆转录酶(货号：18080044)、SuperScriptTMIV逆转录酶(货号：18090200)、凯杰的M-MuLV Reverse Transcriptase(货号：P7040L)。This example uses Universal Reference RNAs (Agilent, catalog number: 740000) to perform the following RNA strand-specific library construction process analysis. Some of the library construction components used in this application are Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) Components of the library preparation kit. The RNA input amount in this example is 10 ng, and the reverse transcriptase tested is NEB's Ultra TM II Directional RNA Library Prep Kit for NEB Next First Strand Synthesis Enzyme Mix, Novozymes' VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Cat. No.: NR605-01) 1st Strand Enzyme Mix 3, Invitrogen TM SuperScript TM II Reverse Transcriptase (Cat. No.: 18064022), SuperScript TM III Reverse Transcriptase (Cat. No.: 18080044), SuperScript TM IV Reverse Transcriptase (Cat. No.: 18090200), Qiagen's M-MuLV Reverse Transcriptase (Cat. No.: P7040L).

1)mRNA的抓取1) Capturing mRNA

根据翌圣生物的HieffmRNA Isolation Master Kit mRNA纯化试剂盒(货号：12603)说明书，进行8个10ng Universal Reference RNAs的mRNA抓取。According to Hieff mRNA Isolation Master Kit mRNA purification kit (Cat. No.: 12603) instructions, to extract mRNA from 8 10ng Universal Reference RNAs.

2)mRNA的片段化2) mRNA fragmentation

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的打断反应缓冲液，进行94℃7min的片段化。Using Hieff from Yisheng Biotechnology The fragmentation reaction buffer of the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) was used for fragmentation at 94°C for 7 min.

3)第一链cDNA的合成3) Synthesis of first-strand cDNA

使用1st cDNA合成反应缓冲液：250mM pH 8.3的Tris-HCl、300mM KCl、20mM DTT、2.5mM dNTPs及凯杰的RNAse Inhibitor(货号：Y9240L)40U/反应进行第一链cDNA的合成。The first-strand cDNA was synthesized using 1st cDNA synthesis reaction buffer: 250mM Tris-HCl, pH 8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs and 40U/reaction of Qiagen's RNAse Inhibitor (Cat. No.: Y9240L) for synthesis.

表1不同逆转录酶的1st cDNA合成反应体系
Table 1 1st cDNA synthesis reaction system of different reverse transcriptases

根据表1配好第一链cDNA合成反应体系，进行第一链cDNA的反应程序：25℃10min；42℃15min；70℃15min；4℃Hold。Prepare the first-strand cDNA synthesis reaction system according to Table 1, and carry out the first-strand cDNA reaction program: 25°C for 10 min; 42°C for 15 min; 70°C for 15 min; 4°C Hold.

4)第二链cDNA的合成4) Synthesis of the second strand cDNA

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version) (货号：12310)建库试剂盒的2nd Reaction Module 2(dUTP)的组分进行含有dUTP的第二链cDNA的合成。Using Hieff from Yisheng Biotechnology Ultima Dual-mode RNA Library Prep Kit(Premixed version) The 2nd Reaction Module 2 (dUTP) component of the library construction kit (Cat. No. 12310) is used to synthesize the second-strand cDNA containing dUTP.

5)接头连接，连接产物的纯化5) Connector ligation and purification of ligation products

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的Ligation Reaction Module的组分，HieffRNA 384CDI Primer for(货号：12414)的PE Adapter对合成好的cDNA进行接头连接。完成连接后，使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)进行0.45x磁珠纯化，然后使用80％乙醇进行2次清洗，最后使用22μL的无菌水进行洗脱。Using Hieff from Yisheng Biotechnology Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) Ligation Reaction Module components, Hieff RNA 384CDI Primer for (Cat. No.: 12414) PE Adapter was used to connect the synthesized cDNA. DNA Selection Beads (a perfect alternative to AMPure XPBeads) (Cat. No. 12601) were used for 0.45x magnetic bead purification, followed by two washes with 80% ethanol and finally elution with 22 μL of sterile water.

6)文库扩增后纯化6) Purification after library amplification

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的2×SuperII High-Fidelity Mix的组分，HieffRNA 384 CDI Primer for(货号：12414)的RP 501及RP 701到RP 707扩增引物，对合成的cDNA接头连接产物进行文库扩增，扩增循环数16个循环，然后使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)进行0.9x磁珠纯化，然后使用80％乙醇进行2次清洗，最后使用30μL的无菌水进行洗脱，然后使用Qubit进行文库定量，文库产量结果如下表2，文库峰图如图2，泳道1-8依次代表表2中实验编号1-8中各个反应所得文库的琼脂糖电泳结果。Using Hieff from Yisheng Biotechnology Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) 2×Super II High-Fidelity Mix components, Hieff RNA 384 CDI Primer for The cDNA adapter-ligated products were amplified using primers RP 501 and RP 701 to RP 707 (Cat. No. 12414) for 16 cycles and then amplified using Hieff DNA Selection Beads magnetic beads (perfect alternative to AMPure XPBeads) (Cat. No. 12601) were used for 0.9x magnetic bead purification, then washed twice with 80% ethanol, and finally eluted with 30 μL of sterile water. The library was then quantified using Qubit. The library yield results are shown in Table 2. The library peak diagram is shown in Figure 2. Lanes 1-8 represent the agarose electrophoresis results of the libraries obtained from each reaction in Experiment No. 1-8 in Table 2.

表2不同逆转录酶的链特建库产量
Table 2 Yield of chain-specific libraries of different reverse transcriptases

使用不同的逆转录酶进行链特建库，发现使用不含放线菌素D的第一链cDNA合成反应缓冲液后，各种逆转录酶建库后产量相差较大，但是文库峰型基本一致。因此，我们进行等质量混库后送测序，分析测序数据。We used different reverse transcriptases to build the chain-specific library and found that the yields of various reverse transcriptases after library construction varied greatly when the first-chain cDNA synthesis reaction buffer without actinomycin D was used, but the library peaks were basically the same. Therefore, we mixed the library of equal quality and sent it for sequencing to analyze the sequencing data.

7)第二代测序分析7) Second generation sequencing analysis

对不同的逆转录酶进行10ng总RNA的mRNA链特异性建库的文库进行测序分析发现。如表3，可以看到，不同的逆转录酶反转录的第一链cDNA建好的文库，常规测序的下机质检指标差异不大；比对到基因组的比例基本都在98％左右，基因检出数目在18000左右，转录本检出数目在45000以上，外显子检出的占比在93％以上；但是对于链特异性建库的重要指标反义链的占比却相差较大，加放线菌素D的试剂盒12310实验编号1的链特异性能到96％以上，其他各个厂家的逆转录酶的链特异性存在差异，这可能与各个酶的DNA为模板的 DNA聚合酶活性(负链转移)有关，具体：在不加放线菌素D的第一链cDNA反应缓冲液中的都存在不同比例的DNA为模板的DNA聚合酶活性(负链转移)，活性最高的是InvitrogenTMSuperScriptTMIV逆转录酶(货号：18090200)，有接近40％的第一链cDNA在反转录中形成了第二链cDNA，其次是诺唯赞的VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina(货号：NR605-01)1st Strand Enzyme Mix 3，有接近30％的第一链cDNA在反转录中形成了第二链cDNA，再次是InvitrogenTMSuperScriptTMIII逆转录酶(货号：18080044)，有接近11％的第一链cDNA在反转录中形成了第二链cDNA，翌圣的1st Strand Enzyme Mix(货号：12309)、InvitrogenTMSuperScriptTMII逆转录酶、NEBNext First Strand Synthesis Enzyme Mix、凯杰M-MuLV Reverse Transcriptase有接近7-8％的第一链cDNA在反转录中形成了第二链cDNA。The sequencing analysis of the mRNA chain-specific library constructed with 10ng of total RNA using different reverse transcriptases revealed that, as shown in Table 3, the quality inspection indicators of the conventional sequencing machine for the libraries constructed using the first-chain cDNA reverse transcribed by different reverse transcriptases were not much different; the proportion of alignment to the genome was basically around 98%, the number of gene detections was around 18,000, the number of transcript detections was over 45,000, and the proportion of exon detections was over 93%; however, the proportion of the antisense chain, an important indicator for chain-specific library construction, varied greatly. The chain-specific performance of the experimental number 1 of the kit 12310 with actinomycin D reached over 96%, and there were differences in the chain specificity of the reverse transcriptases of other manufacturers, which may be related to the DNA template of each enzyme. The activity of DNA polymerase (negative strand transfer) was related to the activity of DNA polymerase (negative strand transfer). Specifically, in the first-strand cDNA reaction buffer without actinomycin D, there were different proportions of DNA as template DNA polymerase activity (negative strand transfer). The most active one was Invitrogen TM SuperScript TM IV Reverse Transcriptase (Cat. No.: 18090200), with nearly 40% of the first-strand cDNA forming the second-strand cDNA during reverse transcription. The second was VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Cat. No.: NR605-01) 1st Strand Enzyme Mix 3 from Novozymes, with nearly 30% of the first-strand cDNA forming the second-strand cDNA during reverse transcription. The third was Invitrogen TM SuperScript TM III Reverse Transcriptase (Cat. No.: 18080044), with nearly 11% of the first-strand cDNA forming the second-strand cDNA during reverse transcription. Yisheng 1st Strand Enzyme Mix (Cat. No.: 12309), Invitrogen TM SuperScript TM Nearly 7-8% of the first-strand cDNA formed the second-strand cDNA during reverse transcription using the NEBNext First Strand Synthesis Enzyme Mix and Qiagen M-MuLV Reverse Transcriptase.

从这个实施例可以给出的启示是：对不同的逆转录酶进行不同的突变改造可以对逆转录酶的这个DNA为模板的DNA聚合酶活性(负链转移)造成显著的影响，如果该活性降低甚至失活，就有可能完全不用再添加放线菌素D来提高链特异性建库的指标。因此，基于这个线索，我们开始对逆转录酶进行改造，筛选完全失活或者具有极低的DNA为模板的DNA聚合酶活性(负链转移)的逆转录酶，用第一链cDNA合成反应液(250mM pH＝8.3的Tris-HCl，300mM KCl，20mM DTT，2.5mM dNTPs)筛选逆转录酶。The inspiration from this example is that different mutations of different reverse transcriptases can have a significant impact on the DNA polymerase activity (negative strand transfer) of the reverse transcriptase with DNA as the template. If the activity is reduced or even inactivated, it is possible that there is no need to add actinomycin D to improve the index of chain-specific library construction. Therefore, based on this clue, we began to transform reverse transcriptases, screen reverse transcriptases that are completely inactivated or have extremely low DNA-templated DNA polymerase activity (negative strand transfer), and use the first-strand cDNA synthesis reaction solution (250mM Tris-HCl, pH=8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs) to screen reverse transcriptases.

表3不同逆转录酶的链特建库后的测序数据

Table 3 Sequencing data after chain-specific library construction with different reverse transcriptases

实施例2：新型逆转录酶的获得Example 2: Acquisition of a novel reverse transcriptase

基于实施例1，我们分析到可以通过改进逆转录酶的DNA为模板的DNA聚合酶活性(负链转移)活性，来达到不添加放线菌素D，做到逆转录酶只有以RNA为模板的DNA聚合酶活性。因此翌圣生物翌圣ZymeEditor平台基于已发表的各种逆转录酶的突变体及文献，寻找抑制DNA为模板的DNA聚合酶活性(负链转移)活性的突变体，确认在鼠白血病病毒的逆转录酶(MMLV逆转录酶)野生型进行了6个位点的定点突变：K152E、L333P、T382A、G504S、D524A、T662A，其氨基酸序列如SEQ ID No：1所示。委托生工生物工程(上海)股份有限公司合成了上述逆转录酶突变体的编码DNA，其序列如SEQ ID No：2所示。将新型逆转录酶的编码基因SEQ ID No：2序列用常规方法进行蛋白表达纯化，得到新型逆转录酶。Based on Example 1, we analyzed that the DNA polymerase activity (negative strand transfer) activity of the reverse transcriptase with DNA as the template can be improved to achieve the DNA polymerase activity with RNA as the template without adding actinomycin D. Therefore, the Yisheng Bio Yisheng ZymeEditor platform is based on the published mutants and literature of various reverse transcriptases to find mutants that inhibit the DNA polymerase activity (negative strand transfer) activity of DNA as the template, and confirm that the wild type of murine leukemia virus reverse transcriptase (MMLV reverse transcriptase) has undergone site-directed mutations at 6 sites: K152E, L333P, T382A, G504S, D524A, T662A, and its amino acid sequence is shown in SEQ ID No: 1. The coding DNA of the above-mentioned reverse transcriptase mutant was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and its sequence is shown in SEQ ID No: 2. The coding gene SEQ ID No: 2 of the new reverse transcriptase was expressed and purified by conventional methods to obtain a new reverse transcriptase.

DDDP(MMLV逆转录酶突变体依赖DNA的DNA聚合酶活性)检测。DDDP (DNA-dependent DNA polymerase activity of MMLV reverse transcriptase mutant) assay.

本申请突变型逆转录酶酶活测定采用焦磷酸定量法，以野生型MMLV逆转录酶为参照。The enzymatic activity of the mutant reverse transcriptase of the present application is determined by the pyrophosphate quantitative method, with the wild-type MMLV reverse transcriptase as a reference.

测定MMLV逆转录酶DDDP活性的原理是以单链环DNA为模板，经MMLV逆转录酶扩增后，产生DNA的同时伴随着焦磷酸的产生。采用焦磷酸试剂盒对产物进行定量时，产生的荧光值与焦磷酸成正比，即所测得的荧光值与DDDP活性成正比。The principle of determining the DDDP activity of MMLV reverse transcriptase is to use single-stranded circular DNA as a template. After amplification by MMLV reverse transcriptase, the production of DNA is accompanied by the production of pyrophosphate. When the product is quantified using a pyrophosphate kit, the fluorescence value produced is proportional to the pyrophosphate, that is, the measured fluorescence value is proportional to the DDDP activity.

焦磷酸检测试剂盒购自AAT Bioquest，MMLV逆转录酶DDDP活性的测试具体实施方式如下：The pyrophosphate detection kit was purchased from AAT Bioquest. The specific implementation method for testing the DDDP activity of MMLV reverse transcriptase is as follows:

(1)将MMLV逆转录酶根据蛋白质浓度稀释成若干浓度梯度；(1) diluting MMLV reverse transcriptase into several concentration gradients according to protein concentration;

(2)根据表4配制引物退火体系。(2) Prepare the primer annealing system according to Table 4.

表4引物退火体系

Table 4 Primer annealing system

在95℃孵育30s，置于冰上静置2min。Incubate at 95°C for 30 seconds and place on ice for 2 minutes.

然后在上述退火反应体系加入待测酶2μL，振荡混匀，在37℃反2min，结束后立即置于冰上终止反应。Then, add 2 μL of the enzyme to be tested to the above annealing reaction system, shake and mix, incubate at 37° C. for 2 min, and immediately place on ice to terminate the reaction.

焦磷酸检测：在96孔酶标板中加入48μL DEPC水，2μL混匀的酶反应液，50μL焦磷酸工作液，酶标仪震荡混匀后，25℃孵育20min后，用酶标仪测量荧光值，根据荧光值计算突变型逆转录酶活力。新型逆转录酶的DDDP活力结果如表5。Pyrophosphate detection: Add 48 μL DEPC water, 2 μL mixed enzyme reaction solution, and 50 μL pyrophosphate working solution to a 96-well ELISA plate. After shaking and mixing with an ELISA reader, incubate at 25°C for 20 minutes, measure the fluorescence value with an ELISA reader, and calculate the activity of the mutant reverse transcriptase based on the fluorescence value. The DDDP activity results of the new reverse transcriptase are shown in Table 5.

表5突变型MMLV逆转录酶的DDDP活力结果
Table 5 DDDP activity results of mutant MMLV reverse transcriptase

从表5可以看出，本申请的新型逆转录酶的DDDP活力能降至野生型的30％及以下，甚至检测不出DDDP活性。It can be seen from Table 5 that the DDDP activity of the novel reverse transcriptase of the present application can be reduced to 30% or less of that of the wild type, and even no DDDP activity can be detected.

实施例3：新型逆转录酶在RNA链特异性建库中的应用Example 3: Application of a novel reverse transcriptase in RNA strand-specific library construction

本实施例是利用Universal Reference RNAs(安捷伦，货号：740000)进行如下所示的RNA链特建库流程分析。使用的部分建库组分是翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的组分。本实施例的RNA投入量为1μg、10ng。具体建库流程参照实施例1的1,5号实验组。12310试剂盒以及新型逆转录酶每个反应的逆转录酶使用量均为200U。This example uses Universal Reference RNAs (Agilent, catalog number: 740000) to perform the following RNA strand-specific library construction process analysis. Some of the library construction components used are Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) library construction kit components. The RNA input amount in this example is 1 μg and 10 ng. The specific library construction process refers to Experimental Groups 1 and 5 of Example 1. The amount of reverse transcriptase used in each reaction of the 12310 kit and the new reverse transcriptase is 200 U.

表6中实验编号1,2&5,6号实验组是使用翌圣HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)；实验编号3,4&7,8除1st cDNA合成反应缓冲液(250mM pH 8.3的Tris-HCl，300mM KCl，20mM DTT，2.5mM dNTPs)不含放线菌素D之外，合成反应缓冲液组分与实验编号1,2&5,6号实验组相同。逆转录酶使用的是新型逆转录酶及凯杰的RNAse Inhibitor(货号：Y9240L)进行第一链cDNA的合成。The experimental groups No. 1, 2, 5, and 6 in Table 6 were treated with Hieff Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310); Experiment No. 3, 4 & 7, 8, except that the 1st cDNA synthesis reaction buffer (250mM Tris-HCl pH 8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs) does not contain actinomycin D, the synthesis reaction buffer components are the same as those of Experiment No. 1, 2 & 5, 6. The reverse transcriptase used was a new reverse transcriptase and Qiagen's RNAse Inhibitor (Cat. No. Y9240L) for the synthesis of the first-strand cDNA.

结合表6，对新型逆转录酶的建库产量及测序数据分析发现，建库产量相当、文库峰型如图3所示基本一致，泳道1-8依次代表表6中实验编号1-8中各个反应所得文库的琼脂糖电泳结果。测序数据的下机质控合格、基本一致，但是在10ng投入量上，链特异性建库质控指标反义链占比相比使用12310试剂盒的5,6号要高2～3％。综上所述，使用新型逆转录酶确实可以实现不使用放线菌D做到10ng-1μg一致的反义链占比，可以达到98％以上。Combined with Table 6, the library construction yield and sequencing data analysis of the new reverse transcriptase found that the library construction yield was equivalent and the library peak shape was basically consistent as shown in Figure 3. Lanes 1-8 represent the agarose electrophoresis results of the libraries obtained from each reaction in experimental number 1-8 in Table 6. The off-machine quality control of the sequencing data was qualified and basically consistent, but at 10ng input, the antisense strand ratio of the chain-specific library construction quality control indicator was 2-3% higher than that of No. 5 and No. 6 using the 12310 kit. In summary, the use of the new reverse transcriptase can indeed achieve a consistent antisense strand ratio of 10ng-1μg without using actinomycetes D, which can reach more than 98%.

表6新型逆转录酶在RNA链特异性建库中的建库产量及测序数据

Table 6 Library yield and sequencing data of novel reverse transcriptase in RNA chain-specific library construction

实施例4：板式RNA链特异性建库试剂盒Example 4: Plate-type RNA strand-specific library construction kit

分别配置以下组分：Configure the following components separately:

组分A：片段化反应组分，采用12310试剂盒中的片段化反应组分；Component A: fragmentation reaction component, using the fragmentation reaction component in the 12310 kit;

组分B：第一链cDNA合成组分，具体为5-25U/μL所述逆转录酶，1-2U/μL RNase抑制剂，150-250mM pH 7-9的Tris-HCl，200-400mM KCl，10-20mM DTT，2.5-15mM dNTPs；Component B: first-strand cDNA synthesis component, specifically 5-25U/μL reverse transcriptase, 1-2U/μL RNase inhibitor, 150-250mM Tris-HCl at pH 7-9, 200-400mM KCl, 10-20mM DTT, 2.5-15mM dNTPs;

组分C：第二链cDNA的合成及末端修复加A组分，具体为0.5-1.5U/μL DNA聚合酶I的大Klenow片段，0.2-1U/μL核糖核酸酶H，0.2-1U/μL T4 DNA聚合酶，0.05-1U/μL Taq DNA聚合酶，0.2-1U/μL T4多聚核苷酸激酶，5-250mM pH7-9的Tris-HCl，20-100mM MgCl2，120-500mM NaCl，0.8-15mM dNTPs，0.8-15mM dUTP，2-100mM ATP；Component C: Synthesis of second strand cDNA and end repair plus component A, specifically 0.5-1.5U/μL large Klenow fragment of DNA polymerase I, 0.2-1U/μL RNase H, 0.2-1U/μL T4 DNA polymerase, 0.05-1U/μL Taq DNA polymerase, 0.2-1U/μL T4 polynucleotide kinase, 5-250mM Tris-HCl at pH 7-9, 20-100mM MgCl 2 , 120-500mM NaCl, 0.8-15mM dNTPs, 0.8-15mM dUTP, 2-100mM ATP;

组分D：接头连接组分，具体为50-500U/μL的T4 DNA连接酶，50-350mM pH7-9的Tris-HCl，10-100mM MgCl2，2-10mM ATP，3-10％v/v的PEG8000。Component D: adapter ligation component, specifically 50-500 U/μL T4 DNA ligase, 50-350 mM Tris-HCl at pH 7-9, 10-100 mM MgCl 2 , 2-10 mM ATP, and 3-10% v/v PEG8000.

组分E：文库扩增组分，采用12310试剂盒中的2×SuperII High-Fidelity Mix的 组分。Component E: Library amplification component, using 2×Super II High-Fidelity Mix Component.

将配置好的各个组分按照反应样本数量加到同一反应板中。Add the configured components to the same reaction plate according to the number of reaction samples.

图1为96T反应的板式RNA链特异性建库试剂盒示意图，板式RNA链特异建库试剂盒是按照图1所示进行分装保存的。其中A组分-E组分分别满足96个反应样本数量；具体A1-H1为12T的A组分；依次类推其他组分。Figure 1 is a schematic diagram of a plate-type RNA strand-specific library construction kit for 96T reactions. The plate-type RNA strand-specific library construction kit is packaged and stored as shown in Figure 1. Components A to E meet the number of 96 reaction samples respectively; specifically, A1-H1 is component A of 12T; and the same applies to other components.

图5为在建库试剂盒的基础上，进一步添加了自动化测序所需的其他反应组分，例如进行第6列加通用接头或者通用扩增引物，本实施例加入的是HieffRNA 384 CDI Primer for(货号：12414)的PE Adapter，第7、8、9列加入的是使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)，分选磁珠的列数可根据需求加入，不限于3列。这种排版可以和自动化完美搭配，不需要再转板，且可以节约客户的排版，节省时间，也可做到不同的自动化平台基本都可以直接使用。FIG5 shows that other reaction components required for automated sequencing are further added to the library construction kit, such as adding a universal adapter or universal amplification primer to column 6. In this example, Hieff RNA 384 CDI Primer for (Item No.: 12414) PE Adapter, the 7th, 8th, and 9th columns are added with Hieff DNA Selection Beads (a perfect alternative to AMPure XPBeads) (Cat. No. 12601), the number of columns of sorting beads can be increased according to demand, not limited to 3 columns. This layout can be perfectly matched with automation, no need to transfer plates, and can save customers' layout and time, and can also be directly used on different automation platforms.

具体的一体化的反应板可以选择常见的不同排布方式的反应板，例如8联排PCR反应板，96孔反应板，当样本数量较多，各组分的预配量较大时，可选择深孔板，以满足反应需求。The specific integrated reaction plate can select common reaction plates with different arrangements, such as 8-row PCR reaction plates and 96-well reaction plates. When the number of samples is large and the pre-mixed amount of each component is large, a deep-well plate can be selected to meet the reaction requirements.

实施例5：板式RNA链特异性建库试剂盒和12310试剂盒的对比测试Example 5: Comparative test of plate-type RNA strand-specific library construction kit and 12310 kit

本实施例是利用Universal Reference RNAs(安捷伦，货号：740000)进行如下所示的板式RNA链特异性建库和12310试剂盒流程分析。使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒。本实施例的RNA投入量为1μg、10ng。This example uses Universal Reference RNAs (Agilent, Catalog No. 740000) to perform the plate-based RNA strand-specific library construction and 12310 kit process analysis as shown below. Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) Library Construction Kit. The RNA input amount in this example is 1 μg, 10 ng.

1)mRNA的抓取1) Capturing mRNA

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的打断反应缓冲液，进行94℃7min的片段化。Using Hieff from Yisheng Biotechnology The fragmentation reaction buffer of the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) was used for fragmentation at 94°C for 7 min.

2)mRNA的片段化2) mRNA fragmentation

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的打断反应缓冲液，进行94℃7min的片段化。Using Hieff from Yisheng Biotechnology The fragmentation reaction buffer of the Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No.: 12310) was used for fragmentation at 94°C for 7 min.

3)第一链cDNA的合成3) Synthesis of first-strand cDNA

板式链特异性建库试剂盒的第一链cDNA合成组分2.0(B组分)包含：25U/μL新型的逆转录酶，2U/μL RNase抑制剂，250mM pH 8.3的Tris-HCl，300mM KCl，20mM DTT，2.5mM dNTPs。根据表7配置第一链cDNA合成反应体系。The first-strand cDNA synthesis component 2.0 (component B) of the plate-type strand-specific library construction kit contains: 25U/μL novel reverse transcriptase, 2U/μL RNase inhibitor, 250mM Tris-HCl, pH 8.3, 300mM KCl, 20mM DTT, 2.5mM dNTPs. Configure the first-strand cDNA synthesis reaction system according to Table 7.

表7试剂盒12310和板式链特异性建库的第一链cDNA合成反应体系

Table 7 First-strand cDNA synthesis reaction system of kit 12310 and plate-type strand-specific library construction

反应条件是：25℃10min，42℃15min，70℃15min，4℃保存。The reaction conditions were: 25°C for 10 min, 42°C for 15 min, 70°C for 15 min, and storage at 4°C.

使用两个试剂盒12310，板式链特异性建库试剂盒完成第一链cDNA的合成。The first-strand cDNA synthesis was completed using two kits: 12310 and Plate-type Strand-Specific Library Construction Kit.

4)第二链cDNA的合成(含有dUTP第二链cDNA的合成)4) Synthesis of the second-strand cDNA (Synthesis of the second-strand cDNA containing dUTP)

板式链特异性建库试剂盒的第二链cDNA合成组份2.0(C组份)包含：1.5U/μL DNA聚合酶I的大Klenow片段，0.25U/μL核糖核酸酶H，0.2U/μL T4 DNA聚合酶，1U/μL Taq DNA聚合酶，1U/μL T4多聚核苷酸激酶，100mM pH 8.0的Tris-HCl，20mM MgCl2，100mM NaCl，0.8mM dNTPs，2mM ATP，0.8mM dUTP。根据表8配置第二链cDNA合成反应体系。The second strand cDNA synthesis component 2.0 (component C) of the plate-type strand-specific library construction kit includes: 1.5 U/μL large Klenow fragment of DNA polymerase I, 0.25 U/μL RNase H, 0.2 U/μL T4 DNA polymerase, 1 U/μL Taq DNA polymerase, 1 U/μL T4 polynucleotide kinase, 100 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 , 100 mM NaCl, 0.8 mM dNTPs, 2 mM ATP, 0.8 mM dUTP. The second strand cDNA synthesis reaction system was configured according to Table 8.

表8试剂盒12310和板式链特异性建库的第二链cDNA合成反应体系
Table 8 Second-strand cDNA synthesis reaction system of kit 12310 and plate-type strand-specific library construction

反应条件是：16℃30min，72℃15min，保存于4℃。The reaction conditions were: 16°C for 30 min, 72°C for 15 min, and stored at 4°C.

使用两个试剂盒12310，板式链特异性建库试剂盒完成第二链含有dUTP的cDNA的合成。Synthesis of the second strand cDNA containing dUTP was completed using two kits 12310, Plate Strand Specific Library Construction Kit.

5)接头连接，连接产物的纯化5) Connector ligation and purification of ligation products

接头连接组分2.0(D组分)包含：500U/μL的Novel T4DNA连接酶，350mM pH8.0的Tris-HCl，16mM MgCl2，2.8mM ATP，10％ PEG8000(v/v)。根据表9配置第cDNA接头连接体系。Adapter Ligation Component 2.0 (Component D) contains: 500 U/μL Novel T4 DNA ligase, 350 mM Tris-HCl at pH 8.0, 16 mM MgCl 2 , 2.8 mM ATP, 10% PEG8000 (v/v). According to Table 9, the cDNA adapter ligation system was prepared.

表9试剂盒12310和板式链特异性建库的cDNA接头连接反应体系

Table 9 cDNA adapter ligation reaction system for kit 12310 and plate-type strand-specific library construction

反应条件：20℃15min；4℃Hold。Reaction conditions: 20℃15min; 4℃Hold.

使用两个试剂盒12310，板式链特异性建库试剂盒完成连接后，使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)进行0.45x磁珠纯化，然后使用80％乙醇进行2次清洗，最后使用22μL的无菌水进行洗脱。After completing the ligation using two kits 12310 and plate-strand specific library construction kit, use Yisheng Hieff DNA Selection Beads magnetic beads (perfect alternative to AMPure XPBeads) (Cat. No. 12601) were used for 0.45x magnetic bead purification, followed by 2 washes with 80% ethanol and finally elution with 22 μL of sterile water.

6)文库扩增后纯化6) Purification after library amplification

使用翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit(Premixed version)(货号：12310)建库试剂盒的2×SuperII High-Fidelity Mix的组分，HieffRNA 384CDIPrimer for(货号：12414)的RP 501及RP 701到RP 707扩增引物，对合成的cDNA接头连接产物进行文库扩增，总投入量1μg RNA时，扩增循环数为10个循环，总投入量10ng RNA时，扩增循环数为16个循环。然后使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)进行0.9x磁珠纯化，然后使用80％乙醇进行2次清洗，最后使用30μL的无菌水进行洗脱，然后使用Qubit进行文库定量，文库产量结果如下表10，文库峰图如图4。Using Hieff from Yisheng Biotechnology Ultima Dual-mode RNA Library Prep Kit (Premixed version) (Cat. No. 12310) 2×Super II High-Fidelity Mix components, Hieff RNA 384CDIPrimer for The cDNA adapter ligation products were amplified using RP 501 and RP 701 to RP 707 amplification primers (Cat. No. 12414). The total input RNA was 1 μg and the number of amplification cycles was 10 cycles. The total input RNA was 10 ng and the number of amplification cycles was 16 cycles. DNA Selection Beads magnetic beads (perfect alternative to AMPure XPBeads) (Cat. No. 12601) were used for 0.9x magnetic bead purification, followed by 2 washes with 80% ethanol, and finally eluted with 30 μL of sterile water. The library was then quantified using Qubit. The library yield results are shown in Table 10 below, and the library peak diagram is shown in Figure 4.

表10试剂盒12310和板式链特异性建库的产量分析
Table 10 Yield analysis of kit 12310 and plate-type chain-specific library construction

使用两个试剂盒12310，板式链特异性建库试剂盒完成1μg、10ng的总RNA的mRNA建库建库，实验编号1&2,5&6是使用全套的12310完成建库，实验编号3&4,7&8是使用板式链特异性建库试剂盒建库。根据表10，可以看到不同投入量，板式链特异性建库试剂盒相比12310产量要略高。因此，我们对上述8个样本进行第二代测序，分析一下测序数据上的差异。Two kits 12310 and plate-strand-specific library construction kit were used to complete the mRNA library construction of 1μg and 10ng of total RNA. Experiment numbers 1&2, 5&6 used the full set of 12310 to complete the library construction, and experiment numbers 3&4, 7&8 used the plate-strand-specific library construction kit to build the library. According to Table 10, it can be seen that the plate-strand-specific library construction kit has a slightly higher yield than 12310 for different input amounts. Therefore, we performed second-generation sequencing on the above 8 samples to analyze the differences in sequencing data.

7)第二代测序分析7) Second generation sequencing analysis

对不同的试剂盒进行的1μg、10ng总RNA的mRNA链特异性建库的文库进行测序分析发现。如表11，可以看到，不同投入量不同试剂盒建库后，等质量的文库混库后送样测序，原始下机数据相差不大，原始数据的下机质控Q20、Q30差异不大；比对到基因组的占比，1μg的总RNA的mRNA建库占比达到98％以上，10ng的总RNA的mRNA建库，板式链特异性建库试剂盒要比12310高1％左右；外显子的占比板式链特异性建库试剂盒要比12310在不同投入量上要略高；链特异性建库的关键质检指标反义链的占比在1μg上相当，都可以达到98.5％以上，在10ng上板式链特异性建库试剂盒要比12310要高，而且板式链特异性建 库试剂盒在不同投入量上存在一致的链特异性。根据上述案例，板式链特异性建库试剂盒在建库性能上是能达到12310的建库产量及测序数据指标，能满足案例1提的假想。The sequencing analysis of the mRNA chain-specific library construction of 1μg and 10ng total RNA using different kits was found. As shown in Table 11, it can be seen that after the library was constructed with different kits with different input amounts, the libraries of equal quality were mixed and sent for sequencing. The original data off the machine were not much different, and the original data off the machine quality control Q20 and Q30 were not much different; the proportion of the mRNA library construction of 1μg total RNA reached more than 98%, and the mRNA library construction of 10ng total RNA, the plate-type chain-specific library construction kit was about 1% higher than 12310; the proportion of exons in the plate-type chain-specific library construction kit was slightly higher than 12310 at different input amounts; the key quality inspection indicator of chain-specific library construction, the proportion of antisense chain, was equivalent at 1μg, both reaching more than 98.5%, and at 10ng, the plate-type chain-specific library construction kit was higher than 12310, and the plate-type chain-specific library construction kit was higher than 12310, and the plate-type chain-specific library construction kit was higher than 12310. The library kit has consistent strand specificity at different input amounts. According to the above case, the plate-type strand-specific library construction kit can achieve a library construction output and sequencing data index of 12310 in terms of library construction performance, which can meet the assumptions mentioned in Case 1.

表11试剂盒12310和板式链特异性建库的不同投入量的文库测序数据
Table 11 Library sequencing data of different input amounts of kit 12310 and plate-type chain-specific library construction

实施例6：板式RNA链特异建库试剂盒的自动化建库测试Example 6: Automated library construction test of plate-type RNA strand-specific library construction kit

本实施例是利用Universal Reference RNAs(安捷伦，货号：740000)、293细胞系总RNA进行如下所示的板式RNA链特异建库的自动化测试。板式RNA链特异建库试剂盒是按照图1所示进行分装保存的。自动化仪使用的MGISP-960高通量自动化样本制备系统。根据翌圣的自动化建库流程操作指导进行自动化建库。因本实施例的板式试剂的排版只占用1-5列(依次对应图5中的A组分到E组分)，6-12列为空孔，所以我们在自动化前准备试剂的时候可以根据图5进行第6列加通用接头或者通用扩增引物，本实施例加入的是HieffRNA 384 CDI Primer for(货号：12414)的PE Adapter(对应图5中的Primer Mix)，第7、8、9列加入的是使用翌圣HieffDNA Selection Beads分选磁珠(完美替代AMPure XPBeads)(货号：12601)(对应图5中的三列DNA纯化磁珠)。这种排版可以和自动化完美搭配，不需要再转板，且可以节约客户的排版，节省时间，也可做到不同的自动化平台基本都可以直接使用。In this embodiment, Universal Reference RNAs (Agilent, catalog number: 740000) and total RNA from 293 cell lines were used to perform automated testing of plate-based RNA strand-specific library construction as shown below. The plate-based RNA strand-specific library construction kit is packaged and stored as shown in Figure 1. The automation instrument uses the MGISP-960 high-throughput automated sample preparation system. Automated library construction was performed according to Yishen's automated library construction process operation instructions. Because the layout of the plate-based reagents in this embodiment only occupies columns 1-5 (corresponding to components A to E in Figure 5, respectively), and columns 6-12 are empty wells, we can add universal adapters or universal amplification primers to column 6 according to Figure 5 when preparing reagents before automation. In this embodiment, Hieff RNA 384 CDI Primer for PE Adapter (Cat. No.: 12414) (corresponding to Primer Mix in Figure 5), the 7th, 8th, and 9th columns are added with Hieff DNA Selection Beads (a perfect replacement for AMPure XPBeads) (Cat. No. 12601) (corresponding to the three rows of DNA purification beads in Figure 5). This layout can be perfectly matched with automation, without the need for plate transfer, and can save customers layout and time, and can also be directly used on different automation platforms.

本实施例的板式链特异建库试剂盒进行了2排自动化测试，A1-A12(mRNA的抓取步骤 中的样本标记号中的12个编号)进行1μg Universal Reference RNAs的mRNA建库，B1-B12(mRNA的抓取步骤中的样本标记号中的12个编号)进行1μg 293总RNA的mRNA建库。将A1-A12的样本添加到板式链特异建库试剂盒的A行中，按照建库反应步骤依次进行反应；将B1-B12的样本添加到板式链特异建库试剂盒的B行中，按照建库反应步骤依次进行反应。The plate-type chain-specific library construction kit of this embodiment was subjected to two rows of automated testing, A1-A12 (mRNA capture step 1 μg Universal Reference RNAs were used to construct mRNA libraries for samples A1-A12 (12 numbers in the sample labeling number in the mRNA capture step), and 1 μg 293 total RNA was used to construct mRNA libraries for samples B1-B12 (12 numbers in the sample labeling number in the mRNA capture step). Samples A1-A12 were added to row A of the plate-type strand-specific library construction kit, and the reactions were carried out in sequence according to the library construction reaction steps; samples B1-B12 were added to row B of the plate-type strand-specific library construction kit, and the reactions were carried out in sequence according to the library construction reaction steps.

表12是A1-A12是Universal Reference RNAs的建库产量及测序分析，表13是B1-B12的293 RNA的建库产量及测序数据分析。通过图6、7对A1-A12到B1-B12文库峰型的分析发现，板式链特异性建库试剂盒重复性很好；表12、13对A1-A12到B1-B12文库产量分析发现，每个孔位的建库产量相当，下机数据的质控都差不多，包括外显子占比、比对到基因组的比例、尤其是链特异性建库指标反义链的占比都相当。通过上述案例可以说明我们的板式的链特异性建库试剂盒是适用于自动化建库。Table 12 shows the library yield and sequencing analysis of Universal Reference RNAs A1-A12, and Table 13 shows the library yield and sequencing data analysis of 293 RNAs B1-B12. Through the analysis of the peak types of A1-A12 to B1-B12 libraries in Figures 6 and 7, it was found that the plate-type strand-specific library construction kit has good repeatability; Tables 12 and 13 show that the library yield of each well position is comparable, and the quality control of the data off the machine is similar, including the proportion of exons, the proportion of alignment to the genome, and especially the proportion of the antisense strand of the strand-specific library construction indicator. The above cases show that our plate-type strand-specific library construction kit is suitable for automated library construction.

表12板式RNA链特异建库试剂盒的自动化A1-A12建库产量及测序数据分析

Table 12 Automated A1-A12 library construction yield and sequencing data analysis of plate-based RNA strand-specific library construction kit

表13板式RNA链特异建库试剂盒的自动化B1-B12建库产量及测序数据分析

Table 13 Automated B1-B12 library construction yield and sequencing data analysis of plate-type RNA strand-specific library construction kit

实施例7：板式RNA链特异性建库试剂盒的稳定性测试Example 7: Stability test of plate-type RNA strand-specific library construction kit

本实施例是利用Universal Reference RNAs(安捷伦，货号：740000)进行如下所示的RNA链特建库的稳定性测试。对于板式RNA链特异性建库试剂盒按照图1所示的形式，分装。进行4℃1周、2周、3周、4周，25℃2天、4天、6天、10天、14天放置后的按照实施例4的板式RNA建库流程，进行1μg建库及测序分析。This example uses Universal Reference RNAs (Agilent, catalog number: 740000) to perform stability tests on RNA chain-specific libraries as shown below. The plate-type RNA chain-specific library construction kit is packaged in the form shown in Figure 1. After being placed at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, and 25°C for 2 days, 4 days, 6 days, 10 days, and 14 days, 1 μg of library construction and sequencing analysis were performed according to the plate-type RNA library construction process of Example 4.

结果如表14所示，可以看出，4℃处理1～4周，25℃处理2～14天，建库产量相比对照-20℃保存，产量变化不大，在±10％的误差以内；文库峰型如图8所示，基本一致；下机数据质检差异不大，比对到基因组的占比相当，基因检出数目及外显子占比也相差不大，链特异性建库测序指标反义链的占比也都维持在98％以上，稳定性较好。The results are shown in Table 14. It can be seen that the library construction yield did not change much compared with the control stored at -20°C after 1 to 4 weeks of treatment at 4°C and 2 to 14 days of treatment at 25°C, and was within an error of ±10%. The library peak shape was basically consistent as shown in Figure 8. There was little difference in the quality inspection of the data off the machine, the proportion of the genome mapped was similar, the number of gene detections and the proportion of exons were also not much different, and the proportion of the antisense chain of the chain-specific library construction sequencing indicator was maintained at more than 98%, with good stability.

表14板式RNA链特建库试剂盒的稳定性建库产量及测序数据

Table 14 Stability of plate-type RNA strand library preparation kit, yield and sequencing data

Claims (20)

一种RNA链特异性建库试剂盒，其包含第一链cDNA合成组分，其中，所述第一链cDNA合成组分至少包括逆转录酶，所述逆转录酶的依赖DNA模板的DNA聚合酶活性在野生型逆转录酶依赖DNA模板的DNA聚合酶活性的30％以下，所述第一链cDNA合成组分中不含放线菌素D。An RNA chain-specific library construction kit comprises a first-chain cDNA synthesis component, wherein the first-chain cDNA synthesis component at least comprises a reverse transcriptase, the DNA template-dependent DNA polymerase activity of the reverse transcriptase is less than 30% of the DNA template-dependent DNA polymerase activity of a wild-type reverse transcriptase, and the first-chain cDNA synthesis component does not contain actinomycin D. 根据权利要求1所述的RNA链特异性建库试剂盒，其中，所述逆转录酶缺失依赖DNA模板的DNA聚合酶活性。The RNA chain-specific library construction kit according to claim 1, wherein the reverse transcriptase lacks DNA template-dependent DNA polymerase activity. 根据权利要求1所述的RNA链特异性建库试剂盒，其中，所述第一链cDNA合成组分包括5-25U/μL所述逆转录酶、1-2U/μL RNase抑制剂、150-250mM pH 7-9的Tris-HCl、200-400mM KCl、10-20mM DTT和2.5-15mM dNTPs。The RNA chain-specific library construction kit according to claim 1, wherein the first-chain cDNA synthesis components include 5-25U/μL of the reverse transcriptase, 1-2U/μL RNase inhibitor, 150-250mM Tris-HCl at pH 7-9, 200-400mM KCl, 10-20mM DTT and 2.5-15mM dNTPs. 根据权利要求1所述的RNA链特异性建库试剂盒，其中，所述RNA链特异性建库试剂盒还包括片段化反应组分、第二链cDNA的合成及末端修复加A组分、接头连接组分和文库扩增组分。The RNA chain-specific library construction kit according to claim 1, wherein the RNA chain-specific library construction kit further comprises a fragmentation reaction component, a second-chain cDNA synthesis and end repair plus A component, an adapter ligation component and a library amplification component. 根据权利要求4所述的RNA链特异性建库试剂盒，其中，所述第二链cDNA的合成及末端修复加A组分包括0.5-1.5U/μL DNA聚合酶I的大Klenow片段、0.2-1U/μL核糖核酸酶H、0.2-1U/μL T4 DNA聚合酶、0.05-1U/μL Taq DNA聚合酶、0.2-1U/μL T4多聚核苷酸激酶、5-250mM pH7-9的Tris-HCl，20-100mM MgCl2、120-500mM NaCl、0.8-15mM dNTPs、0.8-15mM dUTP和2-100mM ATP。The RNA strand-specific library construction kit according to claim 4, wherein the second strand cDNA synthesis and end repair plus component A comprises 0.5-1.5U/μL large Klenow fragment of DNA polymerase I, 0.2-1U/μL ribonuclease H, 0.2-1U/μL T4 DNA polymerase, 0.05-1U/μL Taq DNA polymerase, 0.2-1U/μL T4 polynucleotide kinase, 5-250mM Tris-HCl at pH 7-9, 20-100mM MgCl 2 , 120-500mM NaCl, 0.8-15mM dNTPs, 0.8-15mM dUTP and 2-100mM ATP. 根据权利要求4所述的RNA链特异性建库试剂盒，其中，所述接头连接组分包括50-500U/μL的T4 DNA连接酶、50-350mM pH7-9的Tris-HCl、10-100mM MgCl2、2-10mM ATP和3-10％v/v的PEG8000。The RNA strand-specific library construction kit according to claim 4, wherein the adapter ligation component comprises 50-500 U/μL T4 DNA ligase, 50-350 mM Tris-HCl at pH 7-9, 10-100 mM MgCl 2 , 2-10 mM ATP and 3-10% v/v PEG8000. 根据权利要求1所述的RNA链特异性建库试剂盒，其中，所述逆转录酶为MMLV逆转录酶突变体，所述MMLV逆转录酶突变体选自a1-a2中任一所述的蛋白质：The RNA strand-specific library construction kit according to claim 1, wherein the reverse transcriptase is a MMLV reverse transcriptase mutant, and the MMLV reverse transcriptase mutant is selected from any one of the proteins described in a1-a2: a1：其在野生型MMLV逆转录酶的基础上，发生如下位点中的一个或多个位点的突变：K152E、L333P、T382A、G504S、D524A或T662A；a1: Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A or T662A; a2：与a1的蛋白质具有至少90％的序列同一性，且酶活性及性能与a1的蛋白质基本相当的蛋白质。a2: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1. 根据权利要求7所述的RNA链特异性建库试剂盒，其中，所述逆转录酶为MMLV逆转录酶突变体，所述MMLV逆转录酶突变体的氨基酸序列如SEQ ID NO：1所示。According to the RNA chain-specific library construction kit according to claim 7, wherein the reverse transcriptase is a MMLV reverse transcriptase mutant, and the amino acid sequence of the MMLV reverse transcriptase mutant is shown in SEQ ID NO: 1. 一种双模式转录组快速建库试剂盒，其包含权利要求1-8中任一项所述的RNA链特异性建库试剂盒中的各组分。A dual-mode transcriptome rapid library construction kit, comprising the components of the RNA chain-specific library construction kit according to any one of claims 1 to 8. 一种板式试剂盒，其包括权利要求1-8中任一项所述的RNA链特异性建库试剂盒中的各组分。 A plate-type kit comprising the components of the RNA chain-specific library construction kit according to any one of claims 1 to 8. 根据权利要求10所述的板式试剂盒，其中，所述板式试剂盒包括如下组分：所述片段化反应组分、所述第一链cDNA合成组分、第二链cDNA的合成及末端修复加A组分、所述接头连接组分、和所述文库扩增组分；各组分分别排布在同一反应板中的不同反应孔中。The plate kit according to claim 10, wherein the plate kit comprises the following components: the fragmentation reaction component, the first-chain cDNA synthesis component, the second-chain cDNA synthesis and end repair plus A component, the adapter connection component, and the library amplification component; each component is respectively arranged in different reaction wells in the same reaction plate. 一种自动化测序产品，其包括权利要求10或11所述的板式试剂盒。An automated sequencing product, comprising the plate-type kit according to claim 10 or 11. 一种MMLV逆转录酶突变体，其选自a1-a2中任一所述的蛋白质：A MMLV reverse transcriptase mutant selected from any one of the proteins described in a1-a2: a1：其在野生型MMLV逆转录酶的基础上，发生如下位点中的一个或多个位点的突变：K152E、L333P、T382A、G504S、D524A或T662A；a1: Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A or T662A; a2：与a1的蛋白质具有至少90％的序列同一性，且酶活性及性能与a1的蛋白质基本相当的蛋白质。a2: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1. 根据权利要求13所述的MMLV逆转录酶突变体，其中，所述MMLV逆转录酶突变体的氨基酸序列如SEQ ID NO：1所示。The MMLV reverse transcriptase mutant according to claim 13, wherein the amino acid sequence of the MMLV reverse transcriptase mutant is as shown in SEQ ID NO: 1. 权利要求13或14所述的MMLV逆转录酶突变体的编码基因。A gene encoding the MMLV reverse transcriptase mutant according to claim 13 or 14. 权利要求13或14所述的MMLV逆转录酶突变体在逆转录反应中的应用。Use of the MMLV reverse transcriptase mutant according to claim 13 or 14 in a reverse transcription reaction. 一种RNA链特异性建库方法，其采用依赖DNA模板的DNA聚合酶活性在野生型逆转录酶依赖DNA模板的DNA聚合酶活性的30％以下的逆转录酶，且合成第一链cDNA时不使用放线菌素D。The invention discloses an RNA chain specific library construction method, which adopts a reverse transcriptase whose DNA template-dependent DNA polymerase activity is less than 30% of the DNA template-dependent DNA polymerase activity of a wild-type reverse transcriptase, and does not use actinomycin D when synthesizing the first-chain cDNA. 根据权利要求17所述的RNA链特异性建库方法，其中，所述RNA链特异性建库方法的步骤包括：The RNA chain-specific library construction method according to claim 17, wherein the steps of the RNA chain-specific library construction method include: (1)将mRNA进行片段化处理；(1) fragmenting mRNA; (2)以片段化的mRNA为模板，在逆转录酶的作用下合成第一链cDNA；(2) Using the fragmented mRNA as a template, the first-strand cDNA is synthesized under the action of reverse transcriptase; (3)合成第二链cDNA；(3) Synthesizing the second-strand cDNA; (4)cDNA连接接头，纯化连接产物；以及(4) ligating the cDNA to an adapter and purifying the ligation product; and (5)对cDNA与接头的连接产物进行文库扩增，获得RNA链特异性文库。(5) Amplify the library of the ligation products of cDNA and adapter to obtain an RNA chain-specific library. 根据权利要求17或18所述的RNA链特异性建库方法，其中，所述逆转录酶为MMLV逆转录酶突变体，其选自a1-a2中任一所述的蛋白质：The RNA chain-specific library construction method according to claim 17 or 18, wherein the reverse transcriptase is a MMLV reverse transcriptase mutant selected from any one of the proteins described in a1-a2: a1：其在野生型MMLV逆转录酶的基础上，发生如下位点中的一个或多个位点的突变：K152E、L333P、T382A、G504S、D524A、或T662A；a1: Based on the wild-type MMLV reverse transcriptase, one or more of the following sites are mutated: K152E, L333P, T382A, G504S, D524A, or T662A; a2：与a1的蛋白质具有至少90％的序列同一性，且酶活性及性能与a1的蛋白质基本相当的蛋白质。a2: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1. 根据权利要求19所述的RNA链特异性建库方法，其中，所述逆转录酶为MMLV逆转录酶突变体，其氨基酸序列如SEQ ID NO：1所示。 The RNA chain-specific library construction method according to claim 19, wherein the reverse transcriptase is a MMLV reverse transcriptase mutant, and its amino acid sequence is shown in SEQ ID NO: 1.
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