In-vitro tissue cell nucleus separation method for reducing unicellular amplification bias

Abstract

The invention discloses a single cell sequencing cell nucleus separation method capable of reducing amplification bias and application thereof. The method comprises the following steps: carrying out cracking treatment on a sample to be extracted in a buffer solution II; carrying out primary centrifugal treatment on the cracking product to obtain a cell nucleus precipitate; and washing the cell nucleus precipitate, re-suspending the cell nucleus precipitate in a buffer solution I, carrying out secondary centrifugal treatment, and finally re-suspending and uniformly mixing the cell nucleus precipitate in a buffer solution III to obtain the finally required cell nucleus. According to the method, only conventional and simple reagent consumables are needed, expensive separation equipment, long-time gradient centrifugation and other operations are not needed, and enough and complete single cell nucleuses can be obtained from few tissue samples or cells to be used for single-cell whole-genomeexperimental research. Compared with a method that usesnucleuses processed by a commercial kit, the amplification homogeneity of subsequent whole genome amplification product is better, the amplification effect is similar to that of a complete single cell, and meanwhile time and cost are saved.

Description

technical field

[0001] The invention relates to the field of gene sequencing, and the invention relates to a method for isolating cell nuclei from ex vivo tissues suitable for reducing the bias of single cell amplification. Background technique

[0002] Single-cell sequencing refers to a new technology for high-throughput sequencing analysis of genome, transcriptome, epigenome and other multi-omics at the level of a single cell. The application of improved technology makes it possible to explain the gene structure and gene expression status of individual cells, and to interpret the heterogeneity among cells. Single-cell sequencing can be widely applied to research in developmental biology, immunity, tumors, etc., and a large number of high-quality research and scientific research results have emerged. Among them, single-cell whole-genome sequencing amplifies the whole genome of the DNA of a single cell obtained after dissociation from tumor tissue, and then constructs and s...

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