A method for constructing high-resolution, high-information single-cell hi-c libraries

A cell and library technology, used in chemical libraries, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as not, and achieve the effect of small sample size, improved resolution, and high resolution

Active Publication Date: 2019-10-29
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Therefore, none of the above-mentioned Hi-C methods in the prior art is a practical Hi-C method that can be applied to single cells or a small number of cells.

Method used

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  • A method for constructing high-resolution, high-information single-cell hi-c libraries
  • A method for constructing high-resolution, high-information single-cell hi-c libraries
  • A method for constructing high-resolution, high-information single-cell hi-c libraries

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Embodiment 1

[0088] 1. Sample Processing

[0089] 1.1 Cross-linking

[0090] 1.1.1 Place 1-10 human leukocyte samples in 10 μL of PBS to make cell suspension droplets;

[0091] 1.1.2 Add 10 μL of 4% formaldehyde solution for cross-linking, and let stand at room temperature for 5 minutes;

[0092] 1.1.3 Add 5 μL of 0.25M glycine solution to the reaction droplet to terminate cross-linking. Stand at room temperature for 5 minutes, then ice bath for 15 minutes;

[0093] 1.2 Picking of single cells (a small number of cells)

[0094] Single cells or a small number of cells are picked out by capillary method and placed in lysate.

[0095] Lysis Solution Formula:

[0096] 10mM Tris-HCl pH 7.4

[0097] 30mM NaCl

[0098] 0.2% NP-40

[0099] 10% protease inhibitor cocktail

[0100] sterile water

[0101] 2. Cell Lysis

[0102] 2.1 Microcentrifuge the cell lysis buffer containing the single-cell sample to pool the liquid at the bottom of the tube, and then ice-bath for 45 minutes;

[0103...

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Abstract

The invention provides a method for constructing a high-resolution, abundant-information single-cell Hi-C library. The method has the advantages of a small amount of initial samples, high resolution, a large amount of information, and easy operation. The method for constructing the Hi-C library comprises the following steps: obtaining a small amount of immobilized chromatin; digesting the immobilized chromatin obtained in the step B to obtain immobilized chromatin fragments; directly reconnecting the immobilized chromatin fragments obtained in the step C to obtain reconnected immobilized chromatin fragments; relieving the immobilization of the reconnected immobilized chromatin fragments obtained in the step D to release DNA fragments; amplifying the released DNA fragments in the step E to obtain amplified product; using the amplified product as DNA fragments to be sequenced to construct the DNA library for sequencing.

Description

technical field [0001] The invention relates to a method for constructing a library capable of capturing the three-dimensional conformation of chromatin within the scope of the whole genome, and belongs to the technical field of gene sequencing. Background technique [0002] DNA is the carrier of cell genetic information, exists in each cell in the form of chromatin in organisms, and controls the process of the entire life activity. At present, the vast majority of research on DNA information is carried out by studying the sequence of bases in DNA molecules (one-dimensional information of DNA), and exploring the laws of life activities by analyzing base sequence information. [0003] The nucleus in the real state is a narrow three-dimensional space. DNA with a linear molecular structure will be located in the nucleus in a complex coiled manner. The original one-dimensional DNA sequence is endowed with a three-dimensional spatial conformation, which leads to a large number of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869
Inventor 李小林张介中赵红梅裴志华玄兆伶李大为梁峻彬陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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