Single-cell transcriptome library establishment method and application thereof

Abstract

The invention provides an optimized single-cell transcriptome library establishment method. The method comprises the following steps: (1) obtaining a sample RNA through cell lysis or extraction; (2) performing reverse transcription operation on the sample to make the RNA form cDNA through reverse transcription; and (3) constructing a transcriptome library by using a separated cDNA, wherein the step (3) of constructing the transcriptome library by using the separated cDNA comprises the following steps: (3-1) performing a tail adding reaction or adopting a single-chain connection manner to add an adaptor; (3-2) performing two-chain synthesis on the cDNA and performing an amplification reaction; (3-3) performing exponential amplification; and (3-4) after transcriptome library construction isperformed on the cDNA amplification product subjected to step (3-3) treatment, removing ''useless DNA''; or after the ''useless DNA'' in the cDNA amplification product subjected to step (3-3) treatment, constructing a transcriptome library. Compared with the prior art, the method provided by the invention is more high-efficiency, simpler, more practical and more accurate, has less pollution, lessloss, low costs and good method repeatability, expands the application range of the sample and improves the precision degree of detection.

Description

technical field [0001] The invention relates to the fields of molecular biology and transcriptomics, in particular to a method for establishing a single-cell transcriptome library and its application. Background technique [0002] Transcriptome is the sum of all RNAs that can be transcribed in a living cell, and it is an important means to study cell phenotype and function. [0003] With the development of technology, single-cell RNA sequencing is more and more favored by researchers. Traditional sequencing actually analyzes a cell population. This analysis scheme averages the information of all cells and cannot obtain the difference information of individual cells. If multiple different single cells can be selected from the whole system for study, the whole system can be reconstructed, and this reconstruction process can provide more and more valuable information. In the current research, such as early embryonic development and early cancer, it is difficult to conduct res...

AI Technical Summary

Problems solved by technology

[0009] The existing schemes have the following disadvantages: First, for the currently commonly used single-cell amplification schemes such as SMART-seq, polyA-containing transcripts are enriched by polyT. Since only 2%-60% of mRNAs carry polyA, so This protocol results in the loss of polyA-free transcript information

Second, single-cell transcriptional library building processes (such as REPLI- Single Cell RNA Library Kit, Qiagen) has no rRNA removal step, and about 60%-99% of the information in the library comes from rRNA, this part of the information is of little significance to the current research and will cause a great waste of sequencing data

Finally, in the prior art, in the process of building a library for highly degraded samples such as FFPE, the cost is high due to the need for expensive enzymes and the use of special probes, which limits the application of this technology

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