Method and kit for constructing mRNA chain specific library

文档序号：1486353 发布日期：2020-02-28 浏览：34次中文

阅读说明：本技术 构建mRNA链特异文库的方法及试剂盒 (Method and kit for constructing mRNA chain specific library ) 是由卢超刘江辉马焕班吕艳花赖国荣于 2019-12-03 设计创作，主要内容包括：本公开提供了一种构建mRNA链特异文库的方法,其包括：将mRNA片段、放线菌素D、第一混合酶液和第一缓冲液混合成第一反应体系,使第一反应体系进行第一热处理,获得第一反应液；将第一反应液与第二混合酶液和第二缓冲液混合成第二反应体系,使第二反应体系进行第二热处理,获得第二反应液；将第二反应液与T4DNA连接酶、第三缓冲液和Y字型接头混合成第三反应体系,使第三反应体系进行第三热处理,第三热处理后纯化获得第三反应液；并且在第三反应液中添加预混液、扩增引物和UDG酶混合成第四反应体系,使第四反应体系进行第四热处理,第四热处理后纯化获得mRNA链特异文库。根据本公开能够提供一种有利于提高建库效率的构建mRNA链特异文库的方法及试剂盒。(The present disclosure provides a method of constructing an mRNA strand-specific library, comprising: mixing the mRNA fragment, actinomycin D, the first mixed enzyme solution and the first buffer solution to form a first reaction system, and carrying out first heat treatment on the first reaction system to obtain a first reaction solution; mixing the first reaction solution, the second mixed enzyme solution and the second buffer solution to form a second reaction system, and carrying out second heat treatment on the second reaction system to obtain a second reaction solution; mixing the second reaction solution, T4DNA ligase, a third buffer solution and a Y-shaped joint to form a third reaction system, carrying out third heat treatment on the third reaction system, and purifying after the third heat treatment to obtain a third reaction solution; and adding the premixed solution, the amplification primer and the UDG enzyme into the third reaction solution to mix into a fourth reaction system, carrying out fourth heat treatment on the fourth reaction system, and purifying after the fourth heat treatment to obtain the mRNA chain specific library. According to the method and the kit for constructing the mRNA chain specific library, the library construction efficiency can be improved.)

1. A method of constructing a specific library of mRNA strands, comprising:

the method comprises the following steps:

preparing and mixing an mRNA fragment, actinomycin D, a first mixed enzyme solution having an rnase inhibitor and a reverse transcriptase, and a first buffer solution having a first dNTP as a first reaction system, and subjecting the first reaction system to a first heat treatment in which a first strand of cDNA is synthesized using the mRNA fragment as a template to obtain a first reaction solution, wherein the reverse transcriptase is an MMLV reverse transcriptase lacking in ribonuclease H activity, and the first dNTP is a mixture of dATP, dTTP, dCTP and dGTP;

mixing the first reaction solution with a second mixed enzyme solution containing DNA polymerase I, ribonuclease H, T4, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragments, and a second buffer solution containing a second dNTP, as a second reaction system, and subjecting the second reaction system to a second heat treatment in which a second uracil-containing cDNA strand is synthesized using the cDNA first strand as a template, followed by end repair and end addition a of cDNA composed of the cDNA first strand and the cDNA second strand, to form a second reaction solution, wherein the Klenow fragments lack nuclease activity for gap translation from 5 'end to 3' end and nuclease activity for correction from 3 'end to 5' end, and the second dNTP is a mixture of dATP, dUTP, dCTP and dGTP;

mixing the second reaction solution with T4DNA ligase, a third buffer solution containing dimethyl sulfoxide, and a Y-shaped linker to obtain a third reaction system, subjecting the third reaction system to a third heat treatment in which cDNA is ligated to the Y-shaped linker, and purifying the third heat treatment to obtain a third reaction solution; and is

Adding a premixed solution containing an amplification enzyme, an amplification primer and uracil DNA glycosylase to the third reaction solution, mixing them to form a fourth reaction system, subjecting the fourth reaction system to a fourth heat treatment in which a second uracil-containing cDNA strand is degraded, and then an amplification reaction is performed using the first cDNA strand as a template, followed by purification to obtain an mRNA strand-specific library.

2. The method of claim 1, wherein:

in the first reaction system, the working concentration of actinomycin D is 0.01g/L to 0.05g/L, the working concentration of MMLV reverse transcriptase is 8U/muL to 12U/muL, and the working concentration of RNase inhibitor is 1.0U/muL to 1.5U/muL.

3. The method of claim 1, wherein:

in the second reaction system, the working concentration of the DNA polymerase I is 0.6U/muL to 1U/muL, the working concentration of the glyconuclease H is 0.1U/muL to 0.2U/muL, the working concentration of the T4DNA polymerase is 0.05U/muL to 0.1U/muL, the working concentration of the T4 polynucleotide kinase is 0.3U/muL to 0.5U/muL, the working concentration of the Taq-B DNA polymerase is 0.04U/muL to 0.08U/muL, and the working concentration of the Klenow fragment is 0.08U/muL to 0.12U/muL.

4. The method of claim 1 or 2, wherein:

the first buffer solution also comprises Tris-HCl, magnesium chloride, potassium chloride and dithiothreitol,

in the first reaction system, the working concentration of the first dNTP is 0.3mM to 0.7mM, the working concentration of Tris-HCl is 0.03M to 0.06M, the working concentration of magnesium chloride is 2.0mM to 2.5mM, the working concentration of potassium chloride is 0.03M to 0.08M, and the working concentration of dithiothreitol is 6mM to 10 mM.

5. A method according to claim 1 or 3, characterized by:

the second buffer further comprises Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP;

in the second reaction system, the working concentration of the second dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM.

6. The method of claim 1, wherein:

the mRNA fragments are subjected to a fragmentation treatment, and the fragmentation treatment is carried out in a fragmentation reaction system formed by mixing a fragmentation buffer and an mRNA sample, wherein the fragmentation buffer comprises Tris-HCl, magnesium ions and random hexamer primers.

7. The method of claim 1, wherein:

the third buffer solution also comprises ATP, Tris-HCl, magnesium chloride, dithiothreitol and polyethylene glycol 8000,

in the third reaction system, the working concentration of the T4DNA ligase is 10U/. mu.L to 30U/. mu.L; the working concentration of the Y-shaped joint is 1 x 10 < -7 > M to 3 x 10 < -7 > M; the working concentration of the dimethyl sulfoxide is 1-1.5%, the working concentration of the ATP is 1.5-3 mM, the working concentration of the Tris-HCl is 0.04-0.08M, the working concentration of the magnesium chloride is 0.03-0.05M, the working concentration of the dithiothreitol is 1-3 mM, and the working concentration of the polyethylene glycol 8000 is 5-8 wt%.

8. The method of claim 1 or 7, wherein:

the first sequence of the Y-shaped joint is 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3', the second sequence is 5 '-GATCGGAAGAGCACACGTCTGAACCAGTCACXXXXXATCTCGTATGCCGTCTTCTGCTTG-3',

wherein the 3 'end of the first sequence is modified by thio, the 5' end of the second sequence is modified by phosphorylation, NNNNNNNN is a molecular tag sequence, and XXXXXXX is a sample tag sequence.

9. A kit for constructing an mRNA chain specific library is characterized in that:

the method comprises the following steps: actinomycin D, a first mixed enzyme solution with RNase inhibitor and reverse transcriptase and a first buffer solution with first dNTP, and a second mixed enzyme solution with DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-BDNA polymerase and Klenow fragment and a second buffer solution with second dNTP,

wherein the first dNTP is a mixture of dATP, dTTP, dCTP and dGTP, the second dNTP is a mixture of dATP, dUTP, dCTP and dGTP,

the reverse transcriptase is an MMLV reverse transcriptase lacking ribonuclease H activity, the Klenow fragment lacking nuclease activity for nick translation from 5 'to 3' and nuclease activity for correction from 3 'to 5',

the actinomycin D, the first mixed enzyme solution and the first buffer solution are used for forming a first reaction system for cDNA first strand synthesis, and the second mixed enzyme solution and the second buffer solution are used for forming a second reaction system for cDNA second strand synthesis, end repair and end adding A.

10. The kit of claim 9, wherein:

11. The kit of claim 9 or 10, wherein:

the first buffer solution also comprises Tris-HCl, magnesium chloride, potassium chloride and dithiothreitol,

12. The kit of claim 9 or 10, wherein:

the second buffer further comprises Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP,

13. The kit of claim 9, wherein:

also comprises a fragmentation buffer solution for fragmentation treatment, T4DNA ligase, a third buffer solution with dimethyl sulfoxide and a Y-shaped joint, and a premixed solution with an amplification enzyme, an amplification primer and uracil DNA glycosylase, wherein

The T4DNA ligase, the third buffer solution and the Y-shaped joint are used for forming a third reaction system for connecting the joint, and the premixed solution, the amplification primer and uracil DNA glycosylase are used for forming a fourth reaction system for degrading a uracil-containing cDNA second strand and performing amplification reaction on a cDNA first strand.

Technical Field

The disclosure particularly relates to a method and a kit for constructing an mRNA chain specific library.

Background

The mRNA chain specific library is a sequencing library capable of keeping the direction information of the transcript, the sequence information of the transcript obtained by sequencing is derived from one chain, and the sequence information can be used for determining whether the transcript is derived from a sense chain or an antisense chain, so that the accuracy of the mRNA chain specific library is higher compared with the common transcriptome sequencing library.

At present, the process of constructing an mRNA strand specific library is enrichment of mRNA, then mRNA is fragmented, reverse transcription is performed to synthesize a first strand, dUTP is added to perform second strand synthesis, and then cDNA products are purified, and then steps of double-stranded cDNA end repair and a addition reaction, linker connection, ligation product purification and fragmentation screening, USER enzyme treatment, PCR amplification and purification and the like are performed in sequence.

Disclosure of Invention

The present disclosure has been made in view of the above-described state of the art, and an object thereof is to provide a method and a kit for constructing an mRNA strand-specific library, which are advantageous in improving the efficiency of library construction.

To this end, in one aspect, the present disclosure provides a method of constructing an mRNA strand-specific library, comprising: preparing and mixing an mRNA fragment, actinomycin D, a first mixed enzyme solution having an rnase inhibitor and a reverse transcriptase, and a first buffer solution having a first dNTP as a first reaction system, and subjecting the first reaction system to a first heat treatment in which a first strand of cDNA is synthesized using the mRNA fragment as a template to obtain a first reaction solution, wherein the reverse transcriptase is an MMLV reverse transcriptase lacking in ribonuclease H activity, and the first dNTP is a mixture of dATP, dTTP, dCTP and dGTP; mixing the first reaction solution with a second mixed enzyme solution containing DNA polymerase I, ribonuclease H, T4, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragments, and a second buffer solution containing a second dNTP, as a second reaction system, and subjecting the second reaction system to a second heat treatment in which a second uracil-containing cDNA strand is synthesized using the cDNA first strand as a template, followed by end repair and end addition a of cDNA composed of the cDNA first strand and the cDNA second strand, to form a second reaction solution, wherein the Klenow fragments lack nuclease activity for gap translation from 5 'end to 3' end and nuclease activity for correction from 3 'end to 5' end, and the second dNTP is a mixture of dATP, dUTP, dCTP and dGTP; mixing the second reaction solution with T4DNA ligase, a third buffer solution containing dimethyl sulfoxide, and a Y-shaped linker to obtain a third reaction system, subjecting the third reaction system to a third heat treatment in which cDNA is ligated to the Y-shaped linker, and purifying the third heat treatment to obtain a third reaction solution; and adding a premix solution containing an amplification enzyme, an amplification primer and uracil DNA glycosylase to the third reaction solution, mixing them to form a fourth reaction system, subjecting the fourth reaction system to a fourth heat treatment in which a second uracil-containing cDNA strand is degraded, and then an amplification reaction is performed using the first cDNA strand as a template, followed by purification after the fourth heat treatment to obtain an mRNA strand-specific library.

In the present disclosure, actinomycin D can inhibit the synthesis of the second strand of cDNA during the synthesis of the first strand of cDNA in the first reaction system, thereby contributing to the improvement of strand specificity of an mRNA strand-specific library, rnase inhibitor can prevent mRNA from being degraded, and MMLV reverse transcriptase lacking rnase H activity can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, the second strand cDNA synthesis, end repair and end-plus-A are all carried out in a second reaction system, and in the second reaction system, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and the nuclease activity for nick translation from 5 'end to 3' end and Klenow fragment for correction from 3 'end to 5' end can act synergistically and increase the efficiency of second strand cDNA synthesis, end repair and end-plus-A, thereby contributing to the increase of the efficiency of mRNA strand specific library construction, and the use of a dUTP-containing second dNTP as a starting material can label the second strand cDNA for the subsequent degradation of the second strand cDNA with uracil DNA glycosylase to form an mRNA strand specific library. In addition, the use of the Y-type linker in the linker ligation reaction can improve the ligation efficiency, thereby contributing to the improvement of the efficiency of mRNA library construction.

In addition, in the method for constructing an mRNA chain specific library according to one aspect of the present disclosure, optionally, in the first reaction system, the working concentration of actinomycin D is 0.01g/L to 0.05g/L, the working concentration of MMLV reverse transcriptase is 8U/μ L to 12U/μ L, and the working concentration of RNase inhibitor is 1.0U/μ L to 1.5U/μ L. This can effectively improve the efficiency of first strand cDNA synthesis.

In addition, in the method for constructing an mRNA strand-specific library according to an aspect of the present disclosure, optionally, in the second reaction system, the DNA polymerase I has a working concentration of 0.6U/μ L to 1U/μ L, the glyconuclease H has a working concentration of 0.1U/μ L to 0.2U/μ L, the T4DNA polymerase has a working concentration of 0.05U/μ L to 0.1U/μ L, the T4 polynucleotide kinase has a working concentration of 0.3U/μ L to 0.5U/μ L, the Taq-B DNA polymerase has a working concentration of 0.04U/μ L to 0.08U/μ L, and the Klenow fragment has a working concentration of 0.08U/μ L to 0.12U/μ L. This can effectively improve the reaction efficiency in the second reaction system.

In addition, in the method for constructing an mRNA strand-specific library according to an aspect of the present disclosure, optionally, the first buffer further includes Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and in the first reaction system, the working concentration of the first dNTP is 0.3mM to 0.7mM, the working concentration of the Tris-HCl is 0.03M to 0.06M, the working concentration of the magnesium chloride is 2.0mM to 2.5mM, the working concentration of the potassium chloride is 0.03M to 0.08M, and the working concentration of the dithiothreitol is 6mM to 10 mM. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme.

In addition, in the method for constructing an mRNA strand-specific library according to an aspect of the present disclosure, optionally, the second buffer further includes Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP; in the second reaction system, the working concentration of the second dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme.

In addition, in a method for constructing an mRNA strand-specific library according to an aspect of the present disclosure, optionally, the mRNA fragments are subjected to a fragmentation process, and the fragmentation process is performed in a fragmentation reaction system in which a fragmentation buffer including Tris-HCl, magnesium ions, and random hexamer primers is mixed with an mRNA sample. Thus, a suitable length of mRNA fragment can be obtained and the random hexamer primer required for subsequent synthesis of the first strand of cDNA can be provided.

In addition, in the method for constructing an mRNA strand-specific library according to an aspect of the present disclosure, optionally, the third buffer further includes ATP, Tris-HCl, magnesium chloride, dithiothreitol, and polyethylene glycol 8000, and in the third reaction system, the working concentration of the T4DNA ligase is 10U/. mu.l to 30U/. mu.l; the working concentration of the Y-shaped joint is 1 multiplied by 10^- ⁷M to 3X 10^-7M; the working concentration of the dimethyl sulfoxide is 1-1.5%, the working concentration of the ATP is 1.5-3 mM, the working concentration of the Tris-HCl is 0.04-0.08M, the working concentration of the magnesium chloride is 0.03-0.05M, the working concentration of the dithiothreitol is 1-3 mM, and the working concentration of the polyethylene glycol 8000 is 5-8 wt%. In this case, the third buffer solution can maintain the pH of the third reaction system and the stability of the enzyme, and can enhance the linker ligation reaction.

In addition, in the method for constructing a specific library of mRNA strands according to an aspect of the present disclosure, optionally, the Y-linker has a first sequence of 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3' and a second sequence of 5 '-gatcgaagcacacgtctgaatccagtcacxxxaxatcgtgccgtcttctgcttg-3', wherein the 3 'end of the first sequence is modified by thio, the 5' end of the second sequence is modified by phosphorylation, nnnnnn is a molecular tag sequence and XXXXXXXX is a sample tag sequence. Therefore, the connection efficiency of the Y-shaped joint can be improved, and the accuracy of subsequent sequencing can also be improved.

In another aspect, the present disclosure provides a kit for constructing an mRNA strand specific library, which includes: actinomycin D, a first mixed enzyme solution having an RNase inhibitor and a reverse transcriptase, and a first buffer solution having a first dNTP, and a second mixed enzyme solution having a DNA polymerase I, a ribonuclease H, T4DNA polymerase, a T4 polynucleotide kinase, a Taq-B DNA polymerase, and a Klenow fragment, and a second buffer solution having a second dNTP, wherein the first dNTP is a mixture of dATP, dTTP, dCTP, and dGTP, the second dNTP is a mixture of dATP, dUTP, dCTP, and dGTP, the reverse transcriptase is an MMLV reverse transcriptase lacking ribonuclease H activity, the Klenow fragment lacks nuclease activity for nick translation from 5 'end to 3' end and nuclease activity for correction from 3 'end to 5' end, the actinomycin D, the first mixed enzyme solution, and the first buffer solution are used to form a first reaction system for first strand cDNA synthesis, and the second mixed enzyme solution and the second buffer solution are used to form a second strand synthesis first reaction system for cDNA, And (3) a second reaction system for end repair and end addition of A.

In the present disclosure, actinomycin D can inhibit the synthesis of the second strand of cDNA during the synthesis of the first strand of cDNA, thereby contributing to the improvement of strand specificity of mRNA strand-specific libraries, rnase inhibitors can prevent mRNA from being degraded, MMLV reverse transcriptase lacking rnase H activity can increase the yield of cDNA first strand synthesis, thereby increasing the efficiency of cDNA first strand synthesis. In addition, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-BDNA polymerase and Klenow fragment lacking 5 'to 3' nick translation activity and 3 'to 5' correction can have a synergistic effect, and can improve the efficiency of second strand cDNA synthesis, end repair and end-to-A addition, thus contributing to the improvement of the efficiency of mRNA strand-specific library construction.

In addition, in the kit for constructing an mRNA chain specific library according to another aspect of the present disclosure, optionally, in the first reaction system, the working concentration of actinomycin D is 0.01g/L to 0.05g/L, the working concentration of MMLV reverse transcriptase is 8U/μ L to 12U/μ L, and the working concentration of RNase inhibitor is 1.0U/μ L to 1.5U/μ L; in the second reaction system, the working concentration of the DNA polymerase I is 0.6U/μ L to 1U/μ L, the working concentration of the glyconuclease H is 0.1U/μ L to 0.2U/μ L, the working concentration of the T4DNA polymerase is 0.05U/μ L to 0.1U/μ L, the working concentration of the T4 polynucleotide kinase is 0.3U/μ L to 0.5U/μ L, the working concentration of the Taq-B DNA polymerase is 0.04U/μ L to 0.08U/μ L, and the working concentration of the Klenow fragment is 0.08U/μ L to 0.12U/μ L. This can effectively improve the reaction efficiency of each reaction.

In addition, in a kit for constructing an mRNA strand-specific library according to another aspect of the present disclosure, optionally, the first buffer further includes Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and in the first reaction system, the working concentration of the first dNTP is 0.3mM to 0.7mM, the working concentration of the Tris-HCl is 0.03M to 0.06M, the working concentration of the magnesium chloride is 2.0mM to 2.5mM, the working concentration of the potassium chloride is 0.03M to 0.08M, and the working concentration of the dithiothreitol is 6mM to 10 mM. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme.

In addition, in a kit for constructing an mRNA strand-specific library according to another aspect of the present disclosure, optionally, the second buffer further includes Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP, in the second reaction system, the working concentration of the second dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme.

In addition, in a kit for constructing an mRNA strand-specific library according to another aspect of the present disclosure, optionally, a fragmentation buffer for fragmentation, T4DNA ligase, a third buffer with dimethyl sulfoxide, and a Y-type linker, and a pre-mixture with an amplification enzyme, an amplification primer, and uracil DNA glycosylase, wherein the T4DNA ligase, the third buffer, and the Y-type linker are used to form a third reaction system for linker, and the pre-mixture, the amplification primer, and the uracil DNA glycosylase are used to form a fourth reaction system for degradation of a uracil-containing cDNA second strand and for amplification of a cDNA first strand. Thus, the second uracil-containing cDNA strand can be degraded and the first cDNA strand can be amplified, thereby enabling the amplification of an mRNA strand-specific library.

According to the method and the kit for constructing the mRNA chain specific library, the library construction efficiency can be improved.

Drawings

Figure 1 is a flow diagram illustrating a method of constructing an mRNA strand-specific library according to examples of the present disclosure.

FIG. 2 is a flow diagram showing a method of constructing an mRNA strand-specific library in the comparative example of the present disclosure.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.

In the present disclosure, the unit "M" may be abbreviated as mol/L, the unit "mM" may be abbreviated as mmol/L, and the unit "μ M" may be abbreviated as μmol/L.

Figure 1 is a flow diagram illustrating a method of constructing an mRNA strand-specific library according to examples of the present disclosure.

As shown in fig. 1, the method for constructing an mRNA strand-specific library according to the present embodiment may include: first strand cDNA synthesis (step S10), second strand cDNA synthesis, end repair and end addition of A (step S20), linker ligation (step S30) and library amplification (step S40). In some examples, step S40 may include degradation of the second strand of uracil-containing cDNA.

In some examples, step S10 may include preparing and mixing mRNA fragments, actinomycin D, a first mixed enzyme solution having an rnase inhibitor and a reverse transcriptase, and a first buffer solution having a first dNTP as a first reaction system, and subjecting the first reaction system to a first heat treatment in which a first strand of cDNA is synthesized using the mRNA fragments as a template to obtain the first reaction solution, wherein the reverse transcriptase is an MMLV reverse transcriptase lacking ribonuclease H activity, and the first dNTP is a mixture of dATP, dTTP, dCTP, and dGTP.

In some examples, step S20 may include mixing the first reaction solution with a second mixed enzyme solution having DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment, and a second buffer solution having a second dNTP as a second reaction system, and subjecting the second reaction system to a second heat treatment in which a uracil-containing cDNA second strand is synthesized using the cDNA first strand as a template, followed by end repair and end addition a of cDNA consisting of the cDNA first strand and the cDNA second strand to form the second reaction solution, wherein the low en fragment lacks nuclease activity for gap translation from 5 'end to 3' end and nuclease activity for correction from 3 'end to 5' end, and the second dNTP is a mixture of dATP, dUTP, dCTP and dGTP.

In some examples, the step S30 may include mixing the second reaction solution with T4DNA ligase, a third buffer solution having dimethyl sulfoxide, and a Y-type linker as a third reaction system, and subjecting the third reaction system to a third heat treatment in which cDNA is ligated with the Y-type linker, followed by purification to obtain a third reaction solution.

In some examples, step S40 may include adding a premix solution having an amplification enzyme, an amplification primer, and uracil DNA glycosylase to the third reaction solution to mix as a fourth reaction system, and subjecting the fourth reaction system to a fourth heat treatment in which a second strand of uracil-containing cDNA is degraded, followed by an amplification reaction using the first strand of cDNA as a template, and after the fourth heat treatment, purifying to obtain an mRNA strand-specific library.

In this embodiment, actinomycin D inhibits the synthesis of the second strand of cDNA during the synthesis of the first strand of cDNA, thereby contributing to the improvement of the strand specificity of the mRNA strand-specific library, RNase inhibitors prevent mRNA from being degraded, and MMLV reverse transcriptase with a loss of RNase H activity increases the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis.

In addition, the second strand cDNA synthesis, end repair and end-plus-A are all carried out in a second reaction system, and in the second reaction system, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and the nuclease activity for nick translation from 5 'end to 3' end and Klenow fragment for correction from 3 'end to 5' end can act synergistically and increase the efficiency of second strand cDNA synthesis, end repair and end-plus-A, thereby contributing to the increase of the efficiency of mRNA strand specific library construction, and the use of a dUTP-containing second dNTP as a starting material can label the second strand cDNA for the subsequent degradation of the second strand cDNA with uracil DNA glycosylase to form an mRNA strand specific library.

In addition, the use of the Y-type linker in the linker ligation reaction can improve the ligation efficiency, thereby contributing to the improvement of the efficiency of mRNA library construction. In addition, in the fourth reaction system, uracil dnase can degrade the second strand of cDNA containing uracil, thereby enabling amplification of only the first strand of cDNA with different linker sequences attached to both ends, thereby enabling amplification of an mRNA strand-specific library.

In addition, the second strand synthesis of cDNA, end repair, and end addition of A are all performed in the second reaction system, so that the number of steps (e.g., sample addition, lid closing, machine reaction, lid opening, etc.) for end repair and end addition of A can be reduced, and the purification process after cDNA is formed by second strand synthesis of cDNA can be omitted, whereby the reaction steps can be simplified and the time for library construction can be shortened, and the steps can be omitted, so that the use of reagents can be reduced, and the cost can be reduced.

In some examples, the method of constructing an mRNA strand-specific library may include a preceding step. Among other things, a pre-step can be used to prepare mRNA fragments. Additionally, in some examples, the pre-step may include extracting RNA and isolating mRNA.

In some examples, the sample source from which the RNA is extracted may be a eukaryote. Additionally, in some examples, fungi, blood, paraffin embedded tissue, animal tissue, plant tissue, cultured cells, cell lines, and the like. For example, human renal cortex tissue, mouse liver, young leaves, mature roots, stems, yeast, human oral epithelial cells, and the like.

In some examples, RNA can be extracted and purified using precipitation, column chromatography, or RNA extraction kits to obtain RNA samples. In addition, in some examples, preferably, the RNA integrity value (RIN) of the RNA sample may be greater than 7. In other words, high quality RNA samples are preferred.

In some examples, mRNA samples can be obtained from RNA samples by isolation and purification. In addition, in some examples, mRNA samples can be obtained by capturing ploy a. Thus, an mRNA sample having a poly a tail (ploy a tail) can be isolated from an RNA sample.

In some examples, mRNA may be isolated and purified using oligo (dT) cellulose or oligo (U) agarose affinity chromatography.

In some examples, mRNA samples can be obtained using magnetic beads (i.e., oligo (dt) magnetic beads) with poly-thymidylate (poly-t) modified surfaces. In this case, Poly T on the surface of the magnetic beads can bind to Poly a at the 3' end of mRNA through hydrogen bonding, thereby enabling specific capture of mRNA.

In some examples, mRNA samples can be obtained by an mRNA capture kit. Additionally, in some examples, the mRNA capture kit can include mRNA capture magnetic beads (e.g., oligo (dt) magnetic beads), a magnetic bead binding buffer, a magnetic bead wash, and a Tris buffer.

In some examples, the pre-step may also be a fragmentation process. In addition, in some examples, the mRNA sample may be subjected to a fragmentation process to obtain mRNA fragments (fragmentation reaction solution). In other words, the mRNA fragments may be subjected to a fragmentation process. Therefore, mRNA fragments with proper length can be obtained, and further, the subsequent sequencing of the mRNA chain specific library can be facilitated.

In some examples, the fragmentation process can be performed in a fragmentation reaction system in which a fragmentation buffer is mixed with an mRNA sample. In addition, in some examples, the fragmentation buffer can include Tris-HCl, magnesium ions, and random hexamer primers. In this case, Tris-HCl can be used to maintain the pH stability of the fragmentation reaction system, magnesium ions can be used to break down mRNA, and random hexamer primers required for the subsequent synthesis of the first strand of cDNA can be provided. In addition, a random hexamer primer is added to form a fragmentation buffer solution, and a fragmentation reaction system mixed with the mRNA sample is subjected to fragmentation treatment, so that the random hexamer primer can be better combined with the mRNA fragment.

In some examples, the working concentration of Tris-HCl may be 5mM to 20mM in the fragmentation reaction system. For example, the working concentration of Tris-HCl may be 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM or 20 mM.

In some examples, the working concentration of magnesium ions in the fragmentation reaction system may be 0.3mM to 1.0 mM. For example, the working concentration of magnesium ions may be 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, or 1.0mM, for example.

In some examples, the random hexamer primer can be a mixture of random sequence primers (with 4) comprising 6 bases⁶Seed possible sequence). In addition, in some examples, the 5' end of the random hexamer primer can have a phosphorylation modification.

In some examples, the working concentration of random hexamer primers in the fragmentation reaction system is 30 μ M to 60 μ M. For example, working concentrations of random hexamer primers can be 30. mu.M, 33. mu.M, 35. mu.M, 38. mu.M, 40. mu.M, 43. mu.M, 45. mu.M, 48. mu.M, 50. mu.M, 53. mu.M, 55. mu.M, 58. mu.M, or 60. mu.M.

In some examples, the fragmentation reaction system can perform the fragmentation reaction at 80 ℃ to 95 ℃. This enables cleavage of mRNA by magnesium ions at high temperature. In addition, in some examples, the fragmentation reaction system can perform the fragmentation reaction at 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃ or 95 ℃. In other examples, the fragmentation process may be performed in a PCR instrument.

In some examples, in step S10, the mRNA fragment may be mixed with a first mixed enzyme solution and a first buffer solution into a first reaction system for synthesizing a first strand of cDNA. Specifically, the fragmentation reaction solution may be mixed with a first mixed enzyme solution and a first buffer solution to form a first reaction system for synthesizing a first strand of cDNA.

In some examples, the first mixed enzyme solution may include an rnase inhibitor and a reverse transcriptase. Thus, rnase inhibitors can inhibit mRNA degradation and reverse transcriptase can synthesize the first strand of cDNA. In addition, in some examples, the reverse transcriptase is an MMLV reverse transcriptase lacking rnase H activity. This enables the first strand cDNA to be synthesized more efficiently.

In some examples, the working concentration of MMLV reverse transcriptase in the first reaction system is 8U/. mu.L to 12U/. mu.L and the working concentration of RNase inhibitor is 1U/. mu.L to 1.5U/. mu.L. This can effectively improve the efficiency of first strand cDNA synthesis.

In some examples, the working concentration of MMLV reverse transcriptase in the first reaction system can be 8U/. mu.L, 8.5U/. mu.L, 9U/. mu.L, 9.5U/. mu.L, 10U/. mu.L, 10.5U/. mu.L, 11U/. mu.L, 11.5U/. mu.L, or 12U/. mu.L. In addition, in some examples, the working concentration of the RNase inhibitor in the first reaction system may be 1U/. mu.L, 1.1U/. mu.L, 1.2U/. mu.L, 1.3U/. mu.L, 1.4U/. mu.L, or 1.5U/. mu.L.

In some examples, the first buffer can include a first dNTP. This provides a starting material for first strand cDNA synthesis. In other examples, the first dNTP may refer to a mixture of dATP, dTTP, dCTP, and dGTP. In addition, in some examples, the first dNTP can be an equal mixture of dATP, dTTP, dCTP, and dGTP.

In some examples, the first buffer may also include Tris-HCl, magnesium chloride (MgCl)₂) Potassium chloride (KCl) and Dithiothreitol (DTT). In this case, Tris-HCl is used to maintain the stability of the pH of the first reaction system, magnesium chloride is able to provide magnesium ions to increase the activity of the enzyme, potassium chloride is used to adjust the ionic strength, dithiothreitol is able to maintain the stability of the enzyme, in other words, the first buffer is able to maintain the pH of the first reaction system and the stability of the enzyme, and is able to provide substances (e.g., first dNTP, magnesium ions) and conditions (e.g., pH, ionic strength) required for the reaction.

In some examples, in the first reaction system, the working concentration of the first dNTP may be 0.3mM to 0.7mM, the working concentration of Tris-HCl may be 0.03M to 0.06M, the working concentration of magnesium chloride may be 2.0mM to 2.5mM, the working concentration of potassium chloride may be 0.03M to 0.08M, and the working concentration of dithiothreitol may be 6mM to 10 mM. This provides an environment favorable for first strand cDNA synthesis, and contributes to an improvement in the efficiency of first strand cDNA synthesis.

In some examples, the working concentration of the first dNTP in the first reaction system can be 0.3mM, 0.4mM, 0.5mM, 0.6mM, or 0.7 mM. In addition, in some examples, the working concentration of Tris-HCl may be 0.03M, 0.035M, 0.04M, 0.045M, 0.05M, 0.055M, or 0.06M in the first reaction system.

In some examples, the working concentration of magnesium chloride in the first reaction system may be 2.0mM, 2.1mM, 2.2mM, 2.3mM, 2.4mM, or 2.5 mM. In addition, in some examples, the working concentration of potassium chloride in the first reaction system may be 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M.

In some examples, the working concentration of dithiothreitol in the first reaction system can be 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10 mM.

In some examples, the first reaction system may further comprise actinomycin D. In this case, actinomycin D can inhibit the synthesis of the second strand of cDNA during the synthesis of the first strand of cDNA, and contributes to the improvement of the strand specificity of the mRNA strand-specific library.

In some examples, the working concentration of actinomycin D in the first reaction system may be from 0.01g/L to 0.05 g/L. This can improve the strand specificity of the mRNA strand-specific library.

In some examples, the working concentration of actinomycin D in the first reaction system may be 0.01g/L, 0.015g/L, 0.02g/L, 0.025g/L, 0.03g/L, 0.035g/L, 0.04g/L, 0.045g/L, or 0.05 g/L.

In some examples, the fragmentation reaction is mixed with a first mixed enzyme solution and a first buffer solution in a first reaction system, where the fragmentation reaction may comprise random hexamer primers. In addition, in some examples, the working concentration of random hexamer primers in the first reaction system is 15 μ M to 30 μ M. For example, the working concentration of random hexamer primers can be 15. mu.M, 16. mu.M, 17. mu.M, 18. mu.M, 19. mu.M, 20. mu.M, 21. mu.M, 22. mu.M, 23. mu.M, 24. mu.M, 25. mu.M, 26. mu.M, 27. mu.M, 28. mu.M, 29. mu.M, or 30. mu.M.

In some examples, in step S10, the first reaction system may be subjected to a first heat treatment. Thus, the first reaction system can produce a first strand cDNA synthesis reaction. In other examples, in the first heat treatment, a first strand of cDNA may be synthesized using the mRNA fragment as a template and a first reaction solution obtained. In other examples, the first reaction solution may comprise a first strand of cDNA hybridized to a double strand of mRNA (cDNA-mRNA double strand).

In some examples, the first heat treatment may be performed according to a first predetermined procedure. In other examples, the first heat treatment may be performed in a PCR instrument. Additionally, in some examples, the first predetermined procedure may be: random hexamer primer binding was performed by heating to 25 ℃ for 10min, followed by first strand cDNA synthesis by heating to 42 ℃ for 15min, followed by enzyme inactivation by heating to 70 ℃ for 15 min.

In some examples, in step S20, the first reaction solution may be mixed with a second mixed enzyme solution and a second buffer solution into a second reaction system for synthesizing a second strand of cDNA, end repair, and end-plus-a.

In some examples, the second mixed enzyme solution can include DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragment. In this case, ribonuclease H can remove mRNA before second strand synthesis of cDNA, DNA polymerase I and Klenow fragment can be used for second strand synthesis of cDNA, T4DNA polymerase and T4 polynucleotide kinase can be used for end repair, Taq-B DNA polymerase can be used for end addition of A, and DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment can have a synergistic effect therebetween, which can improve the reaction efficiency of each reaction in the second reaction system.

In some examples, the Klenow fragment can lack nuclease activity for nick translation from 5 'to 3' and nuclease activity for correction from 3 'to 5'.

In some examples, in the second reaction system, the working concentration of DNA polymerase I may be 0.6U/μ L to 1U/μ L, the working concentration of glyconuclease H may be 0.1U/μ L to 0.2U/μ L, the working concentration of T4DNA polymerase may be 0.05U/μ L to 0.1U/μ L, the working concentration of T4 polynucleotide kinase may be 0.3U/μ L to 0.5U/μ L, the working concentration of Taq-BDNA polymerase may be 0.04U/μ L to 0.08U/μ L, and the working concentration of Klenow fragment may be 0.08U/μ L to 0.12U/μ L. This can effectively improve the reaction efficiency in the second reaction system.

In some examples, the working concentration of DNA polymerase I in the second reaction system may be 0.6U/. mu.L, 0.65U/. mu.L, 0.7U/. mu.L, 0.75U/. mu.L, 0.8U/. mu.L, 0.85U/. mu.L, 0.9U/. mu.L, 0.95U/. mu.L, or 1U/. mu.L.

In some examples, the working concentration of glycosylnuclease H in the second reaction system can be 0.1U/. mu.L, 0.11U/. mu.L, 0.12U/. mu.L, 0.13U/. mu.L, 0.14U/. mu.L, 0.15U/. mu.L, 0.16U/. mu.L, 0.17U/. mu.L, 0.18U/. mu.L, 0.19U/. mu.L, or 0.2U/. mu.L.

In some examples, the working concentration of T4DNA polymerase in the second reaction system may be 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, 0.08U/. mu.L, 0.09U/. mu.L, or 0.1U/. mu.L. In addition, in some examples, the working concentration of T4 polynucleotide kinase in the second reaction system can be 0.3U/. mu.L, 0.35U/. mu.L, 0.4U/. mu.L, 0.45U/. mu.L, or 0.5U/. mu.L.

In some examples, the working concentration of Taq-BDNA polymerase in the second reaction system may be 0.04U/. mu.L, 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, or 0.08U/. mu.L. In addition, in some examples, the working concentration of Klenow fragment in the second reaction system can be 0.08U/. mu.L, 0.09U/. mu.L, 0.10U/. mu.L, 0.11U/. mu.L, or 0.12U/. mu.L.

In some examples, the second buffer can include a second dNTP. This provides a starting material for second strand cDNA synthesis. In other examples, the second dNTP can refer to a mixture of dATP, dUTP, dCTP, and dGTP. In addition, in some examples, the second dNTP can be an equal mixture of dATP, dUTP, dCTP, and dGTP.

In particular, the second strand of cDNA synthesized from the second dNTP is base-paired with adenine and uracil, and the first strand of cDNA is base-paired with adenine and thymine, whereby the second strand of cDNA can be distinguished from the first strand of cDNA.

In some examples, the second buffer can further include Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP. In this case, Tris-HCl is used to maintain the stability of pH of the second reaction system, magnesium chloride can provide magnesium ions to increase the activity of the enzyme, sodium chloride is used to adjust the ionic strength, dithiothreitol can maintain the stability of the enzyme, dATP can provide a raw material of terminal addition a, ATP is used to provide energy for the reaction in the second reaction system, in other words, the first buffer can maintain the pH of the first reaction system and the stability of the enzyme, and can provide substances (e.g., second dNTP, magnesium ions, dATP, ATP) and conditions (e.g., pH, ionic strength) required for the reaction.

In some examples, in the second reaction system, the working concentration of the second dNTP may be 0.3mM to 0.6mM, the working concentration of Tris-HCl may be 6mM to 10mM, the working concentration of magnesium chloride may be 0.2mM to 0.5mM, the working concentration of sodium chloride may be 0.03M to 0.06M, the working concentration of dithiothreitol may be 3mM to 6mM, the working concentration of dATP may be 1.2mM to 1.4mM, and the working concentration of ATP may be 0.7mM to 1 mM. This can provide an environment favorable for the reaction in the second reaction system, and can contribute to an improvement in the reaction efficiency of each reaction in the second reaction system. In other examples, the working concentration of dATP can be the working concentration of total dATP comprising dATP in the second dNTP.

In some examples, the working concentration of the second dNTP in the second reaction system can be 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, 0.55mM, or 0.6 mM. In addition, in some examples, the working concentration of Tris-HCl in the second reaction system may be 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10 mM.

In some examples, the working concentration of magnesium chloride in the second reaction system may be 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, or 0.5 mM. In some examples, the working concentration of sodium chloride in the second reaction system may be 0.03M, 0.035M, 0.04M, 0.045M, 0.05M, 0.055M, 0.06M.

In some examples, the working concentration of dithiothreitol in the second reaction system can be 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, or 6 mM. In addition, in some examples, the working concentration of dATP in the second reaction system can be 1.2mM, 1.25mM, 1.3mM, 1.35mM, or 1.4 mM. In other examples, the working concentration of ATP may be 0.7mM, 0.8mM, 0.9mM, or 1mM in the second reaction system.

In some examples, the second reaction system may be subjected to a second heat treatment in step S20. Thus, second strand cDNA synthesis, end repair and end addition of A are carried out in the second reaction system.

In some examples, in the second heat treatment, optionally, a second strand of cDNA is synthesized using the first strand of cDNA as a template, followed by end repair and end addition of a to the cDNA composed of the first strand of cDNA and the second strand of cDNA to form a second reaction solution. In other examples, the second reaction solution may comprise a cDNA fragment consisting of a first strand of cDNA and a second strand of cDNA, end-repaired and end-added with a.

In some examples, the second heat treatment may be performed according to a second predetermined procedure. In other examples, the second heat treatment may be performed in a PCR instrument. Additionally, in some examples, the second predetermined procedure may be heating to 16 ℃ for 30min for second strand cDNA synthesis, end repair, and end addition a.

In some examples, in step S30, the second reaction solution may be mixed with T4DNA ligase, a third buffer solution, and a Y-linker into a third reaction system.

In some examples, the working concentration of T4DNA ligase in the third reaction system may be 10U/. mu.L to 30U/. mu.L. For example, the working concentration of T4DNA ligase may be 10U/. mu.L, 12U/. mu.L, 14U/. mu.L, 15U/. mu.L, 18U/. mu.L, 20U/. mu.L, 22U/. mu.L, 24U/. mu.L, 25U/. mu.L, 26U/. mu.L, 28U/. mu.L, or 30U/. mu.L

In some examples, the working concentration of the Y-linker in the third reaction system may be 1 × 10^-7M to 3X 10^-7And M. For example, the Y-junction may have a working concentration of 1X 10^-7M、1.2×10^-7M、1.4×10^-7M、1.5×10^-7M、1.7×10^-7M、2×10^-7M、2.5×10^-7M、2.7×10^-7M or 3X 10^-7M。

In some examples, the third buffer may include dimethyl sulfoxide (DMSO). This enhances the ligation reaction between the Y-shaped linker and the cDNA. In some examples, the third buffer may also include ATP, Tris-HCl, magnesium chloride, dithiothreitol, and polyethylene glycol 8000(PEG 8000). In this case, Tris-HCl can maintain the pH stability of the third reaction system, magnesium ions in ATP and magnesium chloride are used to catalyze T4DNA ligase, dithiothreitol can maintain the stability of T4DNA ligase, polyethylene glycol 8000 can thicken the third reaction system to facilitate Y-type linker ligation to cDNA, in other words, the third buffer can maintain the pH of the third reaction system and the stability of the enzyme, and can enhance linker ligation reaction.

In some examples, in the third reaction system, the working concentration of dimethyl sulfoxide may be 1% to 1.5%, the working concentration of ATP may be 1.5mM to 3mM, the working concentration of Tris-HCl may be 0.04M to 0.08M, the working concentration of magnesium chloride may be 0.03M to 0.05M, the working concentration of dithiothreitol may be 1mM to 3mM, and the working concentration of polyethylene glycol 8000 may be 5 wt% to 8 wt%. This provides an environment favorable for the linker ligation reaction, and improves the reaction efficiency of the linker ligation reaction.

In some examples, the working concentration of dimethyl sulfoxide in the third reaction system may be 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%. In addition, the working concentration of dimethyl sulfoxide in the third reaction system is expressed in% by volume.

In some examples, the working concentration of ATP may be 1.5mM, 1.8mM, 2mM, 2.3mM, 2.5mM, 2.8mM, or 3mM in the third reaction system.

In some examples, the working concentration of Tris-HCl in the third reaction system may be 0.04M, 0.045M, 0.05M, 0.055M, 0.06M, 0.065M, 0.07M, 0.075M, or 0.08M. In other examples, the working concentration of magnesium chloride in the third reaction system may be 0.03M, 0.035M, 0.04M, 0.045M, or 0.05M.

In some examples, the working concentration of dithiothreitol in the third reaction system can be 1mM, 1.2mM, 1.5mM, 1.8mM, 2mM, 2.2mM, 2.5mM, 2.8mM, or 3 mM.

In some examples, the working concentration of polyethylene glycol 8000 can be 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, or 8 wt% in the third reaction system. In addition, the working concentration of polyethylene glycol 8000 in the third reaction system is expressed in mass percent concentration wt%.

In some examples, the first sequence of the Y-type linker may be 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3' and the second sequence is 5 '-GATCGGAAGAGCACACTCTGTCAACTCCAGTCACXXXXXATCTCGTATGCCGTCTTCTGCTTG-3', thereby improving the efficiency of ligation of the Y-type linker to the cDNA.

In some examples, nnnnnnnnnn may be a molecular tag sequence and XXXXXXXX may be a sample tag sequence. In this case, the molecular tag sequence can be used to identify the cDNA fragment and the sample tag sequence can be used to identify the sample, which can help to improve the accuracy of subsequent sequencing.

In some examples, nnnnnnnnn can be a random base sequence. In other examples, XXXXXXXX can be a random base sequence. In addition, the base sequence of NNNNNNNN can be different from the base sequence of XXXXXXXXX.

In some examples, the 3 'end of the first sequence is modified by thio and the 5' end of the second sequence is modified by phosphorylation. This can further improve the efficiency of ligation of the Y-type linker to cDNA.

In some examples, the third reaction system may be subjected to a third heat treatment in step S30. Thereby, a linker connecting reaction can occur in the third reaction system. In other examples, the cDNA may be ligated to the Y-type linker in the third heat treatment.

In some examples, the third heat treatment may be performed according to a third predetermined program. In other examples, the third heat treatment may be performed in a PCR instrument. Additionally, in some examples, the third predetermined procedure may be heating to 22 ℃ for 15min for joint connection.

In some examples, after the third heat treatment, the third reaction solution may be obtained by subjecting the third reaction system to a purification treatment. In addition, in some examples, the third reaction system after the third heat treatment is purified using magnetic beads. In other examples, the third reaction solution comprises cDNA with a Y-linker attached thereto.

In some examples, in step S40, the third reaction solution may be mixed with the premix and the amplification primers into a fourth reaction system. In other examples, the premix may include an amplification enzyme. In addition, in some examples, the premix may include all of the components necessary to perform PCR, except for the templates and primers.

In some examples, the premix may be a Taq PCR Mix premix, a KAPA HiFi PCR Mix premix, or a Q5 hotspot PCR Mix premix. For example, the premix may be a 1 × KAPA HiFi PCR Mix premix. In other examples, the working concentration of the premix in the fourth reaction system is 1 ×.

In some examples, the amplification primer may be a combination of a forward primer and a reverse primer. In other examples, the sequence of the forward primer may be 5'-AATGATACGGCGACCACCGA-3' and the reverse primer may be 5'-CAAGCAGAAGACGGCATACGA-3'.

In some examples, the working concentration of amplification primers may be 2 × 10^-7M to 6X 10^-7And M. For example, the working concentration of amplification primers may be. 2X 10^-7M、2.5×10^-7M、2.8×10^-7M、3×10^-7M、3.5×10^-7M、4.0×10^-7M、4.5×10^-7M、5.0×10^-7M、5.5×10^-7M、6×10^-7M。

In some examples, the fourth reaction system may further include uracil DNA glycosylase (UNG). Thus, the second strand of cDNA containing uracil can be degraded, thereby being beneficial to constructing an mRNA strand specific library.

In some examples, the working concentration of uracil DNA glycosylase in the fourth reaction system may be 0.02U to 0.08U. Thus, the second strand of uracil-containing cDNA can be efficiently degraded, and the strand specificity of an mRNA strand-specific library can be further improved.

In some examples, the working concentration of uracil DNA glycosylase in the first reaction system can be 0.02U/. mu.L, 0.025U/. mu.L, 0.03U/. mu.L, 0.035U/. mu.L, 0.04U/. mu.L, 0.045U/. mu.L, 0.05U/. mu.L, 0.055U/. mu.L, 0.06U/. mu.L, 0.065U/. mu.L, 0.07U/. mu.L, 0.075U/. mu.L, or 0.08U/. mu.L.

In some examples, in step S40, the fourth reaction system may be subjected to a fourth heat treatment. Thus, the second strand of uracil-containing cDNA can be degraded and an amplification reaction can be carried out in the fourth reaction system. In other examples, in a fourth heat treatment, the second uracil-containing strand of the cDNA may be degraded, and an amplification reaction may be performed using the first cDNA strand as a template.

In some examples, the fourth heat treatment may be performed according to a fourth predetermined program. In other examples, the fourth heat treatment may be performed in a PCR instrument. Additionally, in some examples, the fourth predetermined procedure may include a degradation procedure and a PCR amplification reaction procedure.

In some examples, the degradation procedure may be heating to 98 ℃ for 10 min. L additionally, in some examples, the PCR amplification reaction procedure may be: the pre-denaturation is first carried out by heating to 98 ℃ for 30 seconds, then the denaturation procedure is carried out by maintaining 98 ℃ for 10 seconds, then the annealing procedure is carried out by cooling to 60 ℃ for 30 seconds, then the extension procedure is carried out by heating to 72 ℃ for 30 seconds, and the cycle is 9 to 15 times in the manner of "denaturation → annealing → extension", then the extension is carried out for 5min at 72 ℃ to ensure that the reactants are fully extended. .

In some examples, after the fourth heat treatment, purification of the fourth reaction system may result in an mRNA strand-specific library. In addition, in some examples, the fourth reaction system after the fourth heat treatment is purified using magnetic beads. In other examples, purification may be performed using a PCR product kit.

In some examples, the first reaction system, the second reaction system, and the third reaction system may be formed within one reaction vessel (e.g., reaction tube). This can simplify the operation process. Specifically, after a first reaction system is formed into a first reaction solution by a first heat treatment in a reaction tube, a second mixed enzyme solution and a second buffer solution are continuously added to the reaction tube to form a second reaction system, the second reaction system is formed into a second reaction solution by a second heat treatment in the reaction tube, then T4DNA ligase, a Y-linker and a third buffer solution are continuously added to the reaction tube to form a third reaction system, and then the third reaction system is formed into a third reaction solution by a third heat treatment in the reaction tube.

The kit for constructing an mRNA strand-specific library according to the present embodiment may include: actinomycin D, a first mixed enzyme solution, a first buffer solution, a second mixed enzyme solution and a second buffer solution. In some examples, the first mixed enzyme solution may have an rnase inhibitor and a reverse transcriptase. Wherein the reverse transcriptase is MMLV reverse transcriptase lacking ribonuclease H activity. In other examples, the second mixed enzyme solution can have DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragment. In addition, the Klenow fragment may lack nuclease activity for nick translation from the 5 'end to the 3' end and nuclease activity for correction from the 3 'end to the 5' end. In addition, the kit may include actinomycin D.

In some examples, actinomycin D, the first mixed enzyme solution, and the first buffer solution may be used to form a first reaction system for first strand cDNA synthesis. In other examples, the second mixed enzyme solution and the second buffer solution may be used to form a second reaction system for second strand synthesis, end repair, and end-plus-a of cDNA.

In some examples, the first buffer can include a first dNTP and the second buffer can include a second dNTP. Wherein the first dNTP can be a mixture of dATP, dTTP, dCTP and dGTP, and the second dNTP can be a mixture of dATP, dUTP, dCTP and dGTP.

In this embodiment, rnase inhibitors can prevent mRNA degradation and MMLV reverse transcriptase, which lacks rnase H activity, can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and the lack of 5 'to 3' nick translation nuclease activity and 3 'to 5' correction Klenow fragment can have a synergistic effect, and can improve the second strand synthesis, end repair and end A addition efficiency of cDNA, thus can contribute to the improvement of the mRNA strand specific library construction efficiency

In some examples, optionally, in the first reaction system, the working concentration of actinomycin D is from 0.01g/L to 0.05g/L, the working concentration of MMLV reverse transcriptase is from 8U/μ L to 12U/μ L, and the working concentration of RNase inhibitor is from 1.0U/μ L to 1.5U/μ L; in the second reaction system, the working concentration of DNA polymerase I is 0.6U/μ L to 1U/μ L, the working concentration of glycosylnuclease H is 0.1U/μ L to 0.2U/μ L, the working concentration of T4DNA polymerase is 0.05U/μ L to 0.1U/μ L, the working concentration of T4 polynucleotide kinase is 0.3U/μ L to 0.5U/μ L, the working concentration of Taq-B DNA polymerase is 0.04U/μ L to 0.08U/μ L, and the working concentration of Klenow fragment is 0.08U/μ L to 0.12U/μ L. Therefore, the reaction efficiency of each reaction can be effectively improved.

In some examples, the first buffer may further include Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and the working concentration of dNTPs may be 0.3mM to 0.7mM, Tris-HCl may be 0.03M to 0.06M, magnesium chloride may be 2.0mM to 2.5mM, potassium chloride may be 0.03M to 0.08M, and dithiothreitol may be 6mM to 10mM in the first reaction system. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme, and can provide substances and conditions required for the reaction.

In some examples, the second buffer may further include Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP, and in the second reaction system, the working concentration of dNTP may be from 0.3mM to 0.6mM, the working concentration of Tris-HCl may be from 6mM to 10mM, the working concentration of magnesium chloride may be from 0.2mM to 0.5mM, the working concentration of sodium chloride may be from 0.03M to 0.06M, the working concentration of dithiothreitol may be from 3mM to 6mM, the working concentration of dATP may be from 1.2mM to 1.4mM, and the working concentration of ATP may be from 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme, and can provide substances and conditions required for the reaction.

In some examples, the first mixed enzyme solution, the first buffer solution, and the first reaction system may be as described in the method for constructing an mRNA strand-specific library described above. In other examples, the second mixed enzyme solution, the second buffer solution, and the second reaction system may be as described in the method for constructing an mRNA strand-specific library described above.

In some examples, the kit may include a fragmentation buffer. Among them, a fragmentation buffer may be used for the fragmentation treatment. In addition, in some examples, a fragmentation buffer can be used to form the fragmentation reaction system. Thus, the mRNA fragmentation treatment can be performed using the kit. In other examples, the fragmentation buffer can be as described above in the methods of constructing an mRNA strand-specific library.

In some examples, the kit may include T4DNA ligase, a third buffer, and a Y-linker. In addition, in some examples, T4DNA ligase, a third buffer, and a Y-linker may be used to form a third reaction system. Wherein the third reaction system may be used for ligation linker reactions. In other examples, T4DNA ligase, a third buffer, and a Y-linker may be as described above in the methods for constructing an mRNA strand-specific library.

In some examples, a kit may include a premix, amplification primers, and uracil DNA glycosylase. In other examples, the premix, the amplification primers, and the uracil DNA glycosylase may be used to form a fourth reaction system. In other examples, the premix, amplification primers, uracil DNA glycosylase, and fourth reaction system can be as described above in the methods for constructing an mRNA strand-specific library.

In some examples, the kit may include a fragmentation buffer for a fragmentation process, T4DNA ligase, a third buffer with dimethyl sulfoxide, and a wye-type linker, and a premix with an amplification enzyme, an amplification primer, and a uracil DNA glycosylase, wherein the T4DNA ligase, the third buffer, and the wye-type linker may be used to form a third reaction system for the ligation linker, and the premix, the amplification primer, and the uracil DNA glycosylase may be used to form a fourth reaction system for degrading a uracil-containing cDNA second strand and performing an amplification reaction on the cDNA first strand. Thus, the second uracil-containing cDNA strand can be degraded and the first cDNA strand can be amplified, thereby enabling the amplification of an mRNA strand-specific library.

In some examples, the kit can further include mRNA capture magnetic beads (e.g., oligo (dt) magnetic beads), a magnetic bead binding buffer, a magnetic bead wash, and a Tris buffer. This enables capture of mRNA using the kit.

In some examples, the procedure for constructing an mRNA strand-specific library using the kit may be the same as that of any of the methods described above for constructing an mRNA strand-specific library. In other words, the method for constructing an mRNA chain-specific library using the kit can be the same as any of the above-described methods for constructing an mRNA chain-specific library.

According to the method and the kit for constructing the mRNA chain specific library, the library construction efficiency can be improved.

To further illustrate the present disclosure, the methods for constructing specific libraries of mRNA strands provided by the present disclosure are described in detail below with reference to examples, and the beneficial effects achieved by the present disclosure are fully illustrated with reference to comparative examples.

[ example 1]

In this example, total RNA of purified human leukocyte samples was extracted using a commercial kit as RNA samples, followed by quantification of RNA samples using a qubit3.0 fluorescence quantifier, detection of sample completion by an Agilent 4200 bioanalyzer, and finally selection of RNA samples with a concentration of 216 ng/. mu.l and a RIN of 10 as RNA samples to be processed.

In this example, the formulations of the first buffer solution, the first mixed enzyme solution, the second buffer solution, and the third buffer solution are shown in table 1 below. Wherein the first dNTP is an equal mixture of dATP, dTTP, dCTP and dGTP, and the second dNTP is an equal mixture of dATP, dUTP, dCTP and dGTP. In addition, the first sequence of the Y-junction is 5'-AATGATACGGCGACCACCGAGATCTACACATATGCGCACACTCTTTCCCTACACGACGCTCTTCCGATC-3' and the second sequence is 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTGATCGTATCTCGTATGCCGTCTTCTGCTTG-3'.

TABLE 1

(mRNA isolation and fragmentation)

(1) According to the concentration of the RNA sample of 216 ng/. mu.L, 1000ng of the RNA sample was taken into a 0.2ml PCR tube A, nuclease-free water was then added to a total volume of 50. mu.L and placed on ice for use, 50. mu.L of mRNA capture magnetic beads equilibrated to room temperature were then added, gently pipetted 10 times to mix well, and mRNA capture was performed in a PCR instrument according to the capture procedure shown in Table 2 below.

TABLE 2 Capture procedure

(2) After the program run was complete, PCR tube a was removed and placed on a magnetic stand, and after the solution was clarified, the supernatant was carefully removed.

(3) Taking the PCR tube A out of the magnetic frame, adding 200 mu L of magnetic bead washing liquid to resuspend the magnetic beads, then gently sucking by using a pipettor for 10 times, fully mixing, then placing on the magnetic frame, and carefully removing the supernatant after the solution is clarified.

(4) The PCR tube A was removed from the magnetic frame, 50. mu.L of Tris buffer was added to resuspend the magnetic beads, followed by gentle pipetting 10 times using a pipette and mixing well, followed by mRNA elution in a PCR machine according to the elution procedure shown in Table 3.

TABLE 3 elution procedure

(5) After the program was run, the PCR tube a was removed and 50 μ L of magnetic bead binding buffer was added, followed by gentle pipetting 10 times using a pipette and mixing well, followed by standing at room temperature for 5min to bind mRNA to the magnetic beads.

(6) PCR tube a was placed on a magnetic rack to separate mRNA from total RNA, and after the solution was clarified, the supernatant was carefully removed.

(7) Taking the PCR tube A out of the magnetic frame, adding 200 mu L of magnetic bead washing liquid to resuspend the magnetic beads, then gently sucking by using a pipettor for 10 times, fully mixing uniformly, then placing on the magnetic frame, and carefully removing the supernatant after the solution is clarified.

(8) The PCR tube A was taken out of the magnetic frame, 12. mu.L of fragmentation buffer was added to resuspend the magnetic beads, followed by gentle pipetting 10 times using a pipette and mixing well, and mRNA fragmentation was performed in a PCR instrument according to the fragmentation procedure shown in Table 4.

TABLE 4 fragmentation procedure

(9) After the program run was completed, the PCR tube A was immediately placed on a magnetic stand, after the solution was clarified, 10. mu.L of the supernatant (mRNA fragment) was pipetted into a new PCR tube (i.e., PCR tube B), and a first strand cDNA synthesis reaction was immediately performed.

(cDNA Synthesis, repair and addition of A)

First, in a PCR tube B, a first reaction system for synthesizing a first strand of cDNA is prepared according to the following table 5, a pipette is used for gently sucking and beating for 10 times to fully mix the first strand of cDNA, then a first strand cDNA synthesis reaction is carried out in a PCR instrument according to a first preset program shown in the table 6, wherein the temperature of a PCR hot cover is set to be 105 ℃, and the PCR tube B is immediately taken out after the first preset program is finished running.

TABLE 5 first reaction System

TABLE 6 first predetermined procedure

And (II) preparing a second reaction system for synthesizing a second cDNA chain (containing uracil), repairing the tail end and adding A to the tail end in a PCR tube B according to the table 7, gently sucking and beating the second reaction system for 10 times by using a pipette, fully mixing the second reaction system and the tail end, then reacting in a PCR instrument according to a second preset program shown in the table 8, wherein the temperature of a PCR hot cover is set to be 105 ℃, and immediately taking out the PCR tube B after the second preset program is operated.

TABLE 7 second reaction System

TABLE 8 second Preset procedure

(Joint ligation and purification)

(a) Preparing a third reaction system for synthesizing a connecting joint in the PCR tube B according to the table 9, slightly sucking and beating the third reaction system for 10 times by using a pipette, fully and uniformly mixing the third reaction system and the PCR tube B, and then carrying out joint connection reaction in a PCR instrument according to a third preset program shown in the table 10, wherein the temperature of a PCR hot cover is set to be 105 ℃, and immediately taking out the PCR tube B after the third preset program is finished.

TABLE 9 third reaction System

TABLE 10 third Preset procedure

(b) Add 35. mu.L of water to PCR tube B to make up 100. mu.L, then aspirate 90. mu.L of the mixed magnetic beads (0.9X) and add to PCR tube B, and gently pipette the mixture, incubate at room temperature for 5min, place PCR tube B on a magnetic rack, and remove the supernatant after the solution is clear.

(c) Add 200. mu.L of freshly prepared 80% ethanol to PCR tube B to rinse the beads, incubate at room temperature for 30sec, carefully remove the supernatant.

(d) Repeating step (c) 1 time.

(e) And keeping the PCR tube B on the magnetic frame all the time, and uncapping and drying the magnetic beads at room temperature for about 8 min.

(f) Adding 22 mu L of nuclease-free water into the PCR tube B, gently sucking and beating the mixture by using a pipette, fully and uniformly mixing the mixture, standing the mixture at room temperature for 2min, placing the mixture on a magnetic frame, and carefully sucking 20 mu L of purified product into a new PCR tube (namely the PCR tube C) after the solution is clarified.

(second Strand degradation of cDNA, library amplification and quality control of library)

(i) In the PCR tube C, a fourth reaction system for amplification reaction is prepared according to table 11, and is gently pipetted 10 times to mix well, and then the degradation of the second strand of cDNA containing uracil and the PCR amplification reaction with the first strand of cDNA as a template are performed in the PCR instrument according to a fourth predetermined program shown in table 12, where the temperature of the PCR hot lid is set to 105 ℃, and when the fourth predetermined program is finished, the PCR tube C is immediately taken out.

TABLE 11 fourth reaction System

TABLE 12 fourth Preset procedure

(ii) Pipette 40 μ L of the mixed magnetic beads (0.8 sequence) into PCR tube C, gently pipette, mix, incubate at room temperature for 5min, place the sample on a magnetic rack, and remove the supernatant after the solution is clear.

(iii) Add 200. mu.L of freshly prepared 80% ethanol to PCR tube C to rinse the beads, incubate at room temperature for 30sec, carefully remove the supernatant.

(iv) (iv) repeating step (iii) 1 time.

(v) And keeping the PCR tube C on the magnetic frame all the time, and uncapping and drying the magnetic beads at room temperature for about 8 min.

(vi) And adding 20 mu L of nuclease-free water into the PCR tube C, gently sucking and beating the mixture by using a pipette, fully and uniformly mixing the mixture, standing the mixture at room temperature for 2min, placing the mixture on a magnetic frame, and carefully sucking all supernate into a new PCR tube (namely the PCR tube D) after the solution is clarified to obtain the mRNA chain specific library.

(vii) The concentration of the mRNA chain specific library in the PCR tube D was measured by a Qubit3.0 fluorescence quantitative analyzer, the size of the library was measured by an Agilent 4200 bioanalyzer, and the results of the library quality test are shown in Table 14.

[ examples 2 to 4]

25页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：一种静电纺丝用均匀分散式气流辅助湿度调控系统

Method and kit for constructing mRNA chain specific library

相关技术

网友询问留言