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...

AI Technical Summary

Problems solved by technology

At present, there is no mature and stable method to obtain mononuclear suspension from frozen tissue. At present, there is only a mononuclear suspension separation method for frozen brain tissue through expensive auxiliary equipment, and there is no demonstration of quality control results.

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