Method for building high-flux single-cell full-length transcriptome sequencing library and application of method

Abstract

The application discloses a method for building a high-flux single-cell full-length transcriptome sequencing library and application of the method. The method comprises the following steps: recognizing single cells by adopting a customized micro-hole chip and an ICELL8 platform, and performing cell lysis, mRNA reverse transcription, cDNA pre-amplification and Tn5 transposase library building on the single cells, wherein a product can be directly applied to sequencing; in a process of mRNA reverse transcription or Tn5 transposase library building, introducing a dual-terminal Barcode sequence with a 5' terminal and a 3' terminal in order to obtain a single cell full-length transcriptome. By adopting the method, the single cell recognition flux is high, and the product can be directly appliedto sequencing. When the method is applied to single-cell sequencing, only two days are required for efficiently obtaining thousands of single-cell full-length transcriptome libraries at one time, sothat the labor cost and time cost are lowered greatly; moreover, the reagent cost is lowered greatly through a trace reaction system, and a basis is laid for large-scale single-cell full-length transcript sequencing.

Description

technical field [0001] This application relates to the field of single-cell sequencing, in particular to a method for preparing a high-throughput single-cell full-length transcriptome library and its application. Background technique [0002] In recent years, single-cell sequencing technology has developed rapidly, and important results have been achieved in the fields of developmental research and cancer research. However, high experimental costs are an insurmountable obstacle for researchers. Therefore, high-throughput, low-cost single-cell preparation Technology and sequencing platforms still hold great promise. [0003] Currently commercialized single-cell processing platforms include Fluidigm C1, GemCode from 10×genomics, and ICELL8 from Wafergen. Among them, Fluidigm C1 can identify and determine a single cell by staining cells and taking pictures. This technology is in an integrated chip area, with 48×2 total of 96 tiny microfluidic chambers. Each microfluidic chamb...

AI Technical Summary

Problems solved by technology

ICELL8 uses nanoliter (abbreviated nL) level microwell chip technology, but limited by the capacity of the microwell chip, it can only generate 3' end transcript information; subsequent library construction has low throughput and takes a long time

[0006] Therefore, there is currently no ideal technology that can achieve high-throughput full-length transcript amplification at the single-cell level and at the same time realize integrated sequencing library construction to shorten sequencing time and cost.

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