High-throughput single-cell transcriptome sequencing method and kit

Abstract

The invention discloses a high-throughput single cell transcriptome sequencing method. The method comprises the following steps: capturing and packaging a single cell by using a micro-fluidic chip, carrying out RAN reverse transcription, pre-amplifying cDNA, carrying out primary amplification, carrying out secondary amplification, performing fragmenting, building a library, performing sequencing, conducting analyzing and the like. The sequencing method disclosed by the invention has the advantages of high cell capture efficiency, high flux (up to 10,000 cells), high flexibility ( wherein 1-8 samples can be prepared at the same time), low cross contamination, low double-package rate, simplicity in operation, low cost and the like, and has wide application prospects in various fields such as environment, infectious diseases, rejection after organ transplantation, immunotherapy and the like.

Description

technical field [0001] The invention relates to a gene sequencing method, in particular to a novel high-throughput single-cell transcriptome sequencing method and a corresponding kit, belonging to the field of molecular biology. Background technique [0002] Single-cell transcriptome can detect the heterogeneity of single cells and provide accurate information for the diagnosis and treatment of diseases. Restricted by the processing throughput and cost of single-cell sequencing technology, many large-scale single-cell epigenetics research work cannot be carried out. For example, one of the traditional ways is to use a mouth pipette for micromanipulation, which is low-throughput, time-consuming, and cumbersome; the other way is to use flow cytometry, which requires a large number of samples and requires precision control, damage to cells. The requirements for subsequent database construction are high. [0003] The industry speculates that the combination of microfluidic te...

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Problems solved by technology

[0004] The throughput of the aforementioned C1 system is low, and currently only 938 cells can be produced at most, and the cost is high

The throughput of the aforementioned Microwell-split pool system is also low, usually 1000-2000 cells, the reaction volume is large, the consumption of reagents is high, the cost is high, and the loss of cells is large

In the aforementioned 10X genomics and Biorad systems, the droplet generation device needs to be driven by electricity, which is costly, and each experiment can only do 4 or 8 samples at the same time, and the flexibility is limited.

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