Construction method of single cell transcriptome sequencing library and applications thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses a construction method of a single cell transcriptome sequencing library and applications thereof. The method comprises the following steps: splitting sorted single cells in a pore plate, and carrying out reverse transcription by using a reverse transcription primer to synthesize a first chain cDNA; synthesizing a second chain cDNA through a replacement synthesis reaction; carrying out fragmentation on the double-chain cDNA by using Tn5 transposase; enriching a fragmented template by using PCR; and labeling the enriched fragment by using Index PCR, and performing purification to obtain the single cell transcriptome sequencing library. The Tn5 transposase is used for directly performing enzyme digestion on the double-stranded cDNA, and PCR pre-amplification on the cDNA is not needed, so that the library preference can be reduced, and more accurate transcriptome information can be obtained; and meanwhile, by using the Tn5 transposase, the library establishment efficiency can be improved, and the library establishment time can be shortened. The method is simple in library establishment steps, more genes can be detected, and the proportion of low and medium abundance gene dropout is effectively reduced. Therefore, library establishment time and costs are reduced, and more detailed research on the single cell level is facilitated.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the field of single-cell sequencing, in particular to a method for constructing a single-cell transcriptome sequencing library and its application. Background technique [0002] In the past ten years, RNA sequencing (RNA-seq) technology has developed rapidly and has become an indispensable tool for differential gene expression and mRNA splicing analysis, and has been widely used in the biomedical field. Most of the existing RNA-seq methods are based on cell populations, and the results only reflect the average expression level of genes in multicellular populations, and cannot show the differences in gene expression in each cell. The recently developed single-cell RNA-seq is a new technology for sequencing RNA at the level of a single cell. It can not only effectively resolve the cellular heterogeneity of tissue samples and the transcriptome heterogeneity of cell populations m...

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Problems solved by technology

[0003] At present, the method of single-cell RNA library construction usually requires pre-amplification of a very small amount of RNA in a single cell, such as reverse transcription of RNA into cDNA, and then adding known sequences to both ends of it for PCR amplification, or using IVT technology performs in vitro transcription and amplification of cDNA, but these two library construction methods cannot avoid the bias introduced by PCR amplification, resulting in problems such as uneven transcriptome coverage, large background noise, and inaccurate quantification; similarly, these methods will Due to the loss of some transcripts during the amplification and capture process, gene dropout is caused, especially for genes with low and medium expression levels, the dropout phenomenon will be more obvious, and these genes with low and medium expression levels are important for analyzing the co-occurrence between genes and genes. expression, and the contribution of medium and low expressed genes to biological pathways is very important

Although most single-cell transcriptome sequencing methods such as SCRB-Seq and Drop-Seq can detect a large number of cells, they can only sequence the 3′ end of the transcript, and each cell can only detect a small number of genes ;Smart-Seq method can use template switching to amplify full-length cDNA, which increases the number of genes detected, but the number of detected cells is small, the steps are complicated, and the cost is higher compared to other methods

In addition, traditional sonication and enzyme fragmentation library construction methods require complicated steps such as end repair and adapter addition to realize library construction.

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