Method and kit for constructing mRNA chain specific library

Abstract

The invention provides a method for constructing an mRNA chain specific library, and the method comprises the following steps: mixing an mRNA fragment, actinomycin D, a first mixed enzyme solution anda 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 with a second mixed enzyme solution and a 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 with T4DNA ligase, a third buffer solution and a Y-shaped linker 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 a premixed solution, an amplification primer and UDG enzyme into the third reaction solution, mixing to form 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 thepresent invention, the method and the kit for mRNA chain specific library construction can be provided, wherein the mRNA chain specific library construction method and the kit can easily improve thelibrary construction efficiency.

Description

technical field [0001] The present disclosure particularly relates to a method and a kit for constructing an mRNA strand-specific library. Background technique [0002] The mRNA strand-specific library is a sequencing library that can retain the orientation information of transcripts. The transcript sequence information obtained by sequencing is derived from one strand, which can be used to determine whether the transcript is derived from the sense strand or the antisense strand. Therefore, unlike ordinary transcription Compared with group sequencing libraries, the accuracy of mRNA strand-specific libraries is higher. [0003] At present, the process of constructing an mRNA strand-specific library is to enrich mRNA, then fragment the mRNA, reverse transcribe to synthesize the first strand, add dUTP to synthesize the second strand, and then purify the cDNA product, followed by double Strand cDNA end repair and A reaction, adapter ligation, ligation product purification and f...

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Problems solved by technology

[0003] At present, the process of constructing an mRNA strand-specific library is to enrich mRNA, then fragment the mRNA, reverse transcribe to synthesize the first strand, add dUTP to synthesize the second strand, and then purify the cDNA product, followed by double Strand cDNA end repair and A reaction, adapter ligation, ligation product purification and fragmentation screening, USER enzyme treatment, PCR amplification and purification, etc. However, this construction process has problems such as complicated steps, long time-consuming, and low library construction efficiency

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