Assay for transposase accessible chromatin using sequencing (ATAC-seq) method applied to zebrafish embryos

Abstract

The invention relates to an assay for transposase accessible chromatin using sequencing (ATAC-seq) method applied to zebrafish embryos. The method comprises the following steps: firstly, performing zebrafish embryo sample pretreatment; adding lysis buffer into the embryos; cracking the embryos in a water bath by using a wild-neck spearhead; performing a transposition reaction immediately and purifying DNA (Deoxyribose Nucleic Acid); determining a cycle number for library establishment by using a real-time fluorescence quantification method; directly performing library establishment and sequencing by using a further PCR (Polymerase Chain Reaction) method. A regulatory sequence on genome chromatin can be located and decoded by the ATAC-seq method and a DNase-seq (DNase I hypersensitive sites sequencing) method, but the steps in ATAC-seq are simpler, a required cell quantity is smaller, the ATAC-seq is more helpful under the situation that a large quantity of cells cannot be obtained, and the data enriching degree of the ATAC-seq is higher according to an assay result. The ATAC-seq method is further applied to zebrafish embryo cells. Thus, an effective and simple method is provided for the researches of changes of chromatin structures in early developing processes of living organisms and corresponding gene expression regulation and control researches.

Description

technical field [0001] The invention belongs to the technical field of molecular biology experiments, and is especially suitable for the research on the change of chromatin structure and the regulation mechanism of gene expression during the early embryonic development of zebrafish. Background technique [0002] There is growing evidence that many genetic differences do not affect genes, but instead alter the regulatory sequences that turn genes on and off. The activated regulatory sequences are bound by transcription factors, which are proteins that recognize specific DNA sequences. Once a transcription factor binds, it recruits other proteins, enabling transcription of nearby genes to begin. Identification of all active regulatory sequences in a given cell is important for understanding the mechanism of action of the genome. Assay for Transposase Accessible Chromatinusingsequencing (ATAC-seq), through Tn5 transposase, preferentially label and sequence DNA between nucleos...

AI Technical Summary

Problems solved by technology

[0004] At present, ATAC-seq technology is only used in cell samples, and it has not been reported to be applied in zebrafish embryonic cells