Preparation method of fish vascular sac single cell sequencing sample

Abstract

The invention discloses a preparation method of a fish vascular sac single cell sequencing sample, which comprises the following steps: rinsing a complete fish vascular sac obtained by separation with a buffer solution, placing the material in a sterile filter screen, dropwise adding the buffer solution or a culture medium to wrap the vascular sac, kneading and pressing the vascular sac, extruding and screening cells in the fish vascular sac, and performing freeze drying to obtain the single-cell sequencing sample of the fish vascular sac; preparing the obtained cells into a single-cell suspension, and filtering the single-cell suspension through a screen with the mesh diameter of not more than 40 microns to obtain a fish vascular sac single-cell suspension which is used as a fish vascular sac single-cell sequencing sample. The method provided by the invention can greatly reduce the cell sieving damage, reduce the generation of cell debris, separate out the complete vascular capsule adventitia, and reduce the connective tissue interference in the single cell suspension. After the single-cell suspension is obtained, the single-cell suspension is filtered through a mesh sieve with the pore diameter being smaller than 40 micrometers, impurities such as redundant cell masses and connective tissues are removed again; the content of the cell masses and the impurities in the single-cell suspension is greatly reduced after the single-cell suspension is sieved twice, and therefore, the complete single-cell suspension almost free of the cell masses and the cell debris can be obtained.

Description

technical field [0001] The invention relates to a method for culturing fish cells, in particular to a method for preparing a fish vascular sac single-cell sequencing sample. [0002] technical background [0003] The DNA that is traditionally sequenced is derived from a large number of cells mixed in a tissue, and the result is only an average of this population of cells. However, there is heterogeneity between cells, and even if the phenotype is the same, the genetic information of cells may have significant differences. Single-cell sequencing technology can not only measure gene expression levels in cells more accurately, but also detect rare non-coding RNA and trace gene expressors. At the same time, by sequencing a variety of cells, the transcriptome map of single cells can be obtained, and cells can be classified and new cells can be defined based on gene expression, the spatial organization of cells and their molecules can be mapped, and the complex gene network of cel...

AI Technical Summary

Problems solved by technology

The preparation of single-cell suspension is the key to single-cell sequencing. It is difficult to prepare single-cell suspension without cell clumps and impurities, and the cell survival rate is low, which greatly reduces the need for gel-in-oil beads (GEM) for subsequent sequencing. ), and the cost of preparing a sample of gel beads (GEM) is more than 10,000 yuan. In the case of low success rate, the cost of single-cell sequencing is very high. Therefore, it is necessary to establish a single-cell sequencing standard. The preparation method of the single-cell sequencing sample of the replicated fish vascular sac provides an experimental platform for the study of the molecular mechanism of fish

Images