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Method for preparing chromatin co-immunoprecipitation library by using trace of clinical puncture sample and application of method

A co-immunoprecipitation and chromatin technology, applied in the biological field, can solve the problems of low success rate, cumbersome steps, and complicated chromatin co-immunoprecipitation experimental steps.

Inactive Publication Date: 2020-07-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the traditional ChIP-seq technology has the advantages of high resolution, low noise, and high coverage, there are still some problems: First, the current ChIP-seq requires too much sample input, and the total amount of tissue samples needs to be at least 200mg Second, the chromatin immunoprecipitation experiment steps are cumbersome in the early stage, requiring formaldehyde cross-linking, cell lysis, ultrasonic disruption, antibody enrichment of target protein and DNA complexes, target protein digestion and cross-linking, purification of target DNA fragments, and DNA fragment end repair, target DNA fragment end adenylation, target DNA fragment connection adapter, PCR amplification and other steps
The cumbersomeness of the steps leads to defects such as long library construction cycle and low success rate

Method used

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  • Method for preparing chromatin co-immunoprecipitation library by using trace of clinical puncture sample and application of method
  • Method for preparing chromatin co-immunoprecipitation library by using trace of clinical puncture sample and application of method
  • Method for preparing chromatin co-immunoprecipitation library by using trace of clinical puncture sample and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Example 1. Preparation method of chromatin immunoprecipitation library

[0079] The flow chart of the rapid co-immunoprecipitation based on the Tn5 transposition system of the present invention is as follows figure 1 shown. These include clinical puncture sample collection, formaldehyde cross-linking, tissue homogenization, cell lysis, ultrasonic interruption of DNA fragments, target protein immunoprecipitation, Tn5 transposition system to break the DNA fragments bound by the target protein and one-step ligation of library adapters, Protein digestion and decompression of DNA fragments bound by cross-linked target proteins, enrichment of DNA fragments bound by target proteins and amplification. Specific steps are as follows:

[0080] 1. Put the clinical puncture sample tissue into a 1.5ml centrifuge tube, add 500μl pre-cooled PBS solution (pH 7.4) to wash the tissue, use a desktop centrifuge at 4°C and 500g for 10 minutes, remove the PBS solution, and get Washed tissu...

Embodiment 2

[0099] Example 2, Application of the preparation method of chromatin immunoprecipitation library

[0100] 1. Clinical puncture samples

[0101] Liver biopsy samples from 3 different individuals (from the Second Affiliated Hospital of Nanchang University, with informed consent from all individuals) were used as clinical biopsy samples, and the initial volumes of clinical biopsy samples were 7.7 mg, 11.4 mg, and 5.5 mg, respectively.

[0102] 2. Preparation of chromatin immunoprecipitation library

[0103] Based on the clinical biopsy samples in step 1, a chromatin immunoprecipitation library was constructed according to the method in Example 1. Wherein, the target protein is H3K4me3, and the specific target protein antibody in step 6 is H3K4me3 antibody (ActiveMotif). The sequences of the upstream and downstream primers in step 12 are as follows (NNNNNNNNNN is the library Index sequence):

[0104] S501:

[0105] AATGATACGGCGACCACCGAGATCTACANNNNNNTCGTCGGCAGCGTC;

[0106] N7...

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Abstract

The invention discloses a method for preparing a chromatin co-immunoprecipitation library by using a trace clinical puncture sample and an application of the method. The invention successfully develops the method for preparing the chromatin co-immunoprecipitation sequencing library based on trace of clinical puncture sample by utilizing high-sensitivity Tn5 transposase, and ChIP-seq library construction can be effectively and rapidly carried out on the trace of puncture tissue sample. Compared with a traditional ChIP-seq library building mode, the method has the advantages that the initial sample amount is greatly reduced (5-50 mg), the library building time is effectively shortened (about 1.5 days), and the requirements of the sequencing market on trace samples and large-scale sample processing speed are met.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a chromatin immunoco-precipitation library using a small amount of clinical puncture samples and an application thereof. Background technique [0002] Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a revolutionary technology that combines chromatin immunoprecipitation (ChIP) with high-throughput sequencing. This technique can identify binding sites on chromatin, such as transcription factors, histone modifications, nucleosomes, and RNA-binding proteins, on a genome-wide scale. Different from the traditional chip-based ChIP-chip (chromatin immunoprecipitation combined with DNA tiling arrays), ChIP-seq provides a new strategy for studying protein-DNA interactions with high resolution, high specificity, and high coverage. ChIP-seq technology can be applied to any species whose genome sequence is known, can study the interaction bet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12Q1/6869C12N15/10C40B50/06
CPCC12Q1/6804C12Q1/6869C12N15/1093C40B50/06
Inventor 薛愿超苏瑞宝王磊
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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