A kind of ultrasonic fragmentation method of pig adipose tissue and its application

Abstract

The invention belongs to the field of chromatin immunoprecipitation, and specifically relates to a method for ultrasonically disrupting porcine adipose tissue and its application, including (1) carrying out formaldehyde cross-linking reaction on ice for porcine adipose tissue, (2) centrifuging after terminating cross-linking with glycine on ice And ice bath stratification, (3) obtaining porcine adipose tissue cell sedimentation layer, (4) obtaining cell nucleus precipitation layer, (5) ultrasonic disruption of chromatin to obtain small fragments of protein-nucleic acid complex five steps. The ultrasonic crushing method of porcine adipose tissue of the present invention maintains low temperature conditions throughout the experimental operation, which ensures the natural structure of the protein to a greater extent, thereby obtaining the DNA-protein complex adsorbed by the real antibody. And the obtained small fragments of the protein-nucleic acid complex can be directly used for subsequent ChIP, the concentration of the obtained DNA is detected, and the combination of the DNA and the target protein in the whole genome is detected by high-throughput sequencing. And further use the enriched dna to build a library and apply it to the bioinformatics analysis of porcine adipose tissue DNA after next-generation sequencing.

Description

technical field [0001] The invention belongs to the field of chromatin immunocoprecipitation, and in particular relates to a method for ultrasonically disrupting porcine adipose tissue and its application. Background technique [0002] The regulation of biological processes in living organisms is very complex and orderly. Most of the genetic information of living organisms exists in the chromatin of cells, and in the biological activities of living organisms, the interaction between protein and DNA is an important basis for the precise regulation of cells. At present, the commonly used methods for detecting DNA-protein interaction in vitro include footprinting method, electrophoretic mobility shift analysis, DNA-Western blotting, yeast one-hybrid analysis, phage display technology, etc. (Dey B, Thukral S, Krishnan S, Chakrobarty M , Gupta S, Manghani C, Rani V. DNA-protein interaction: methods for detection and analysis. Molecular and Cellular Biochemistry. 08 Mar 2012, 365...

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Problems solved by technology

[0004] The pretreatment method of tissue samples in ChIP technology is mainly to cut the tissue samples into pieces, mix them with enzymes such as collagenase and hyaluronidase, digest them at 37°C for 3 hours, and centrifuge the filtrate obtained by conventional formaldehyde fixation and ultrasonic crushing. When this method is used to deal with adipose tissue, the experimental results are very unsatisfactory

Adipose tissue contains a large amount of fat, which makes it difficult for small adipose tissue to precipitate during centrifugation, and the standard ChIP operation procedure requires the use of SDS lysis buffer as the buffer for sonication, but the presence of a large amount of fat seriously affects the sonication efficiency, resulting in The results of chromatin fragmentation are not suitable for subsequent co-immunoprecipitation; and during the enzymatic digestion process of co-immunoprecipitation experiments at 37°C, due to the instability of the protein structure, it is easy to cause partial protein degradation and affect the experimental results

[0005] At present, most studies on adipose ChIP-seq are conducted in preadipocytes, and the dynamic changes of chromatin modification and the relationship with gene expression regulation are also limited to the cellular level, and there is no report on porcine adipose tissue. application

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