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Method for preparing small-amount sample high-throughput chromatin co-immunoprecipitation sequencing sample

A co-immunoprecipitation and sample preparation technology, which is applied in the field of sample preparation for high-throughput chromatin immunoprecipitation sequencing of a small number of samples, can solve the problems of a large number of initial samples and low throughput, and achieve the effect of low price and simple processing technology

Active Publication Date: 2021-10-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to solve the problem of a large number of initial samples and low throughput required by traditional ChIP-seq experiments by combining the ChIP-seq sample preparation process with a microfluidic chip

Method used

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  • Method for preparing small-amount sample high-throughput chromatin co-immunoprecipitation sequencing sample
  • Method for preparing small-amount sample high-throughput chromatin co-immunoprecipitation sequencing sample
  • Method for preparing small-amount sample high-throughput chromatin co-immunoprecipitation sequencing sample

Examples

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Effect test

Embodiment 1

[0086] Example 1. Sample preparation process based on the novel superhydrophobic microwell array chip high-throughput chromatin immunoprecipitation-sequencing (ChIP-seq) technology

[0087] First of all, the processing of the new super-hydrophobic microhole array chip is carried out, as follows:

[0088] 1) PMMA mold design. The mold material is PMMA, and there are 108 microhole arrays (12×9) designed on the mold. The microholes are cylindrical, and the microholes are evenly distributed in the microhole array. is 2.25mm. A superhydrophobic material coating pool is designed between the micropore arrays, the micropores and the micropore wall periphery are all the coating pools, and the depth of the coating pool is 0.1mm. The micropore wall thickness is 0.15mm.

[0089] 2) Synthesis of novel superhydrophobic coatings. 2g 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (Sigma, 718467) was mixed with 198g absolute ethanol for 2 hours under mechanical stirring, and 10g of titanium ...

Embodiment 2

[0103] The specific application example of embodiment 2, embodiment 1 method

[0104] 1. Using the method in Example 1, the H3K4me3 ChIP-seq of 100 samples and 1000 K562 cells in each sample is completed based on the chip. Since each microwell is independently coded, it can be split after sequencing Samples with different encodings were obtained, and compared with the K562 cell H3K4me3 ChIP-seq results in the Encode database, there was good consistency. For specific results, see Figure 6 .

[0105] 2. Adopt the method in Example 1 to implement four different histone modifications based on the chip (H3K4me1 (Abcam, ab8895); H3K4me3 (Millipore, 07-473); H3K27ac (Abcam, ab4729) and H3K27me3 (Millipore, 07-449 )) for ChIP-seq sample preparation with 24 microwell replicates for each histone modification, with an initial volume of approximately 1000 K562 cells per sample. For specific results, see Figure 7 . in, Figure 7 Middle a is a schematic diagram of the seeding distri...

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Abstract

The invention discloses a method for preparing a small-amount sample high-throughput chromatin co-immunoprecipitation sequencing sample. The method comprises on-chip steps of conducting cell inoculation, cross-linking, cell lysis, chromatin fragmentation, specific co-immunoprecipitation reaction, in-situ DNA library construction and high-throughput sample coding; and off-chip steps of recovering an encoded sample from a chip, and then carrying out non-specific cleaning, de-crosslinking, purification, pre-amplification and on-chip sequencing. The chip is a novel super-hydrophobic micropore array chip. A high-throughput chromatin co-immunoprecipitation-sequencing (ChIP-seq) technical platform established on the basis of the novel super-hydrophobic microporous array chip provided by the invention can provide a convenient and feasible solution for realizing a ChIP-seq technology of a small number of cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing high-throughput chromatin immunoprecipitation sequencing samples for a small number of samples. Background technique [0002] Epigenetics is the study of changes in gene expression levels without changes in the gene sequence, which are often heritable. The research content of epigenetics includes: DNA methylation, histone modification, non-coding RNA and chromatin remodeling, etc. Among them, histone modification is an important content in the study of epigenetics. [0003] As the basic structural unit of chromatin, nucleosome is composed of octamer structure of histone and a DNA fragment of about 147bp wound on it, and there are multiple amino acid residues at the N-terminal of histone, such as arginine residues A variety of chemical modifications such as methylation, acetylation, and phosphorylation can occur on the base and lysine residues. These modifica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2565/113C12Q2565/629C12Q2565/631C12Q2535/122C12Q2525/191
Inventor 刘鹏陈忠尧胡亚伟
Owner TSINGHUA UNIV
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