High-throughput single cell transcriptome sequencing method and application thereof

Abstract

The invention provides a high-throughput single-cell transcriptome sequencing method and application thereof. The method comprises the following steps: preparing a to-be-detected cell sample into a single-cell suspension, and fixing the single-cell suspension with a fixing solution; performing in-situ reverse transcription reaction on the RNA of the fixed single cell by using a reverse transcription primer to synthesize a first cDNA chain; wherein the reverse transcription primer comprises a tag sequence.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the field of single-cell sequencing, in particular to a single-cell transcriptome sequencing method for multiple types of cells and applications thereof. Background technique [0002] Currently the most widely used high-throughput single-cell transcriptome sequencing technology is the single-cell sequencing technology based on droplet microfluidic platform developed by 10X Genomics, which can realize the labeling, sequencing and analysis of thousands of cells, and obtain single-cell sequencing technology. The gene expression profile at the cell level can realize the division of cell subgroups and the detection of differentially expressed genes between cell subgroups. Similar technologies include inDrop technology and Drop-seq technology. [0003] The basic operation and principles of 10X Genomics transcriptome sequencing technology are (see figure 1 ): prepare the sample into a single...

AI Technical Summary

Problems solved by technology

[0005] The reverse transcription primers used in the high-throughput single-cell transcriptome sequencing technology represented by 10X Genomics are all Poly(dT) reverse transcription primers, so only part of the transcript information at the 3' end can be obtained, and undesired transcripts cannot be detected. RNA containing polyA (including damaged mRNA fragments and polyA-free miRNA, lncRNA, etc.), which leads to low sensitivity of this technology in practical applications, usually less than 10% of mRNA can be detected

At the same time, this technology has high requirements on RNA quality. If you want to obtain better sequencing results, the cell viability of the sequencing sample is greater than 80%, and the sequencing effect is often poor when using frozen or fixed samples.

In addition, because bacterial RNA does not carry polyA, existing high-throughput single-cell transcriptome sequencing technologies cannot be used for bacterial transcriptome sequencing

At the same time, in order to avoid contamination in this technology, it is necessary to make each droplet include only one cell as much as possible when separating cells. For this reason, the amount of droplets is much higher than the amount of cells in the preparation process and usually only One type of single cell is sequenced, which results in only about 1 / 10 of the droplets having cells in the end, and the beads and reagents in the large number of empty droplets are wasted

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