MRNA library construction method and kit

Abstract

The invention provides an mRNA library construction method. The mRNA library construction method includes mixing an mRNA fragment with a first mixed enzyme solution and a first buffer solution to obtain a first reaction system, and subjecting the first reaction system to first heat treatment to obtain a first reaction solution; mixing the first reaction solution with a second mixed enzyme solutionand a second buffer solution to obtain a second reaction system, and subjecting the second reaction system to second heat treatment to obtain a second reaction solution; mixing the second reaction solution with T4DNA ligase, a third buffer solution and a Y-shaped joint to obtain a third reaction system, subjecting the third reaction system to third heat treatment, and after the third heat treatment, performing purification to obtain a third reaction solution; adding a pre-mixed solution and a amplification primer into the third reaction solution to obtain a fourth reaction system, subjectingthe fourth reaction system to fourth heat treatment, and after the fourth heat treatment, performing purification to obtain an mRNA library. The mRNA library construction method and a kit, which are beneficial to improvement of the library construction efficiency, can be provided.

Description

technical field [0001] The present disclosure particularly relates to a method and a kit for constructing an mRNA library. Background technique [0002] Transcriptome sequencing (RNA-seq) is a sequencing technology that uses next-generation sequencing technology to comprehensively and rapidly obtain the sequence information and expression information of almost all transcripts in a specific cell or tissue in a certain state. Based on different research directions and strategies, transcriptome sequencing is divided into mRNA-seq for coding proteins and Ribo-zero RNA-seq for various non-coding RNAs. [0003] At present, the main process of constructing an mRNA-seq library includes enrichment of mRNA, and then fragmentation of mRNA, reverse transcription of one strand and two strands to synthesize double strands (cDNA at 16°C, 1h), and purification of cDNA products ( About 30min), followed by double-stranded cDNA end repair and A reaction (20°C, 15min; 65°C, 15min), adapter lig...

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Problems solved by technology

[0003] At present, the main process of constructing an mRNA-seq library includes enrichment of mRNA, and then fragmentation of mRNA, reverse transcription of one strand and two strands to synthesize double strands (cDNA at 16°C, 1h), and purification of cDNA products ( About 30min), followed by double-stranded cDNA end repair and A reaction (20°C, 15min; 65°C, 15min), adapter ligation, ligation product purification and fragmentation screening, PCR amplification and purification and other steps, however, this The construction process has problems such as complicated and time-consuming steps and low library construction efficiency

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