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A high-resolution conformation technique of genome-wide chromatin based on a small number of cells ehi-c 2.0

A chromatin and cell technology, applied in recombinant DNA technology, combinatorial chemistry, nucleotide library, etc., can solve the problems of poor data coverage and low interaction resolution.

Active Publication Date: 2021-06-15
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult for these existing technologies to strike a balance between the comprehensiveness of the data and the amount of experimental materials; while the data coverage obtained by the newly emerging single-cell Hi-C technology is poor, and it can only partially show that the gene interaction is relatively small. Strong domains, low resolution genome-wide interactions obtained data

Method used

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  • A high-resolution conformation technique of genome-wide chromatin based on a small number of cells ehi-c 2.0
  • A high-resolution conformation technique of genome-wide chromatin based on a small number of cells ehi-c 2.0
  • A high-resolution conformation technique of genome-wide chromatin based on a small number of cells ehi-c 2.0

Examples

Experimental program
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Effect test

Embodiment 1

[0085] Example 1. Preparation of eHi-C 2.0 sequencing library based on a small number of cells and its application in Hi-C sequencing

[0086] figure 1 It is a flowchart of high-efficiency and high-resolution eHi-C 2.0 sequencing based on 0.1 million cells.

[0087] 1. Lysing cells to prepare chromatin ( figure 2 Optimize processing of major steps for stepwise lysis of cells. )

[0088] 1. Cell collection and cross-linking fixation

[0089] 1) When the cell confluency in the culture dish (10CM) reaches 80-90%, the C2C12 cells ( CRL-1772 TM ) about 6-8million (accurate result of the improved bovine Bauer cytometer);

[0090] 2) Add 37% formaldehyde aqueous solution (280 ul) in mass percentage to it to make the final concentration 1%, put the petri dish on a rotator and shake it at room temperature for 10 minutes (or incubate at 37° C. for 10 minutes);

[0091] 3) Add 2.5M glycine aqueous solution (0.89ml, so that the final concentration is 0.2M) to quench the formaldeh...

Embodiment 2

[0175] Embodiment 2, the restriction exonuclease combination selection test that eliminates linearized DNA

[0176] In Step 5 of Example 1, Lambda and Exonuclease I were used to eliminate the linearized DNA in the circular DNA mixture. The present invention did the following restriction exonuclease combination selection experiment when selecting the combination of these two enzymes for digestion, using PGL -4.23 Plasmids are tested as circular DNA mixtures:

[0177] 1. Acquisition of PGL-4.23 plasmid: Escherichia coli was used to transform, shake the bacteria, and extract the plasmid to obtain a plasmid with a concentration of 110ng / ul.

[0178] 2. Acquisition of linear pGL4.23-1: pGL4.23-1 is the pGL4.23 plasmid (Promega E8411) which was digested with BamHI. The system is as follows: plasmid 23.4ul; BamHI 5ul; 10×Cutsmart Buffer (NEB B7204s) 20ul; H 2 O151.6ul; Total 200ul. Digested at 37°C for 1 hour, purified with 1.2 times phenol chloroform after digestion, and the conce...

Embodiment 3

[0189] Example 3, Enzyme Combination Screening Test for Digesting Chromatin

[0190] In step 2 of Example 1, MboI and HpyCH4IV were used to digest chromatin, and the present invention did the following enzyme combination selection experiment when selecting these two enzyme combinations for enzyme digestion:

[0191] 1. Take 6.6million cells / tube, add to 660ul, suck 10ul (that is, 0.1million) into ten 1.5EP tubes with Ruining white pipette tip, and carry out the first and second operations of Example 1, but in the second 3 ) step enzyme digestion time is divided into the following groups:

[0192] MboI single enzyme digestion group, add 2ul MboI (5U / ul, NEB R0147L);

[0193] HpyCH4IV single enzyme digestion group: add 2ul HpyCH4IV (10U / ul, NEB R0619L);

[0194] MboI and HpyCH4IV double restriction group (M&H): add 1ul MboI (5U / ul, NEB R0147L) and 1ul (10U / ul, NEB R0619L) of HpyCH4IV;

[0195] Each of the above groups was digested for 0h, 4h and 8h respectively (such as ima...

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Abstract

The invention discloses eHi-C 2.0, a high-resolution conformation technology based on a small number of cells and whole genome chromatin. The invention provides a preparation method of a cell eHi-C 2.0 sequencing library: lyse cells to obtain chromatin, and then digest The chromatin; the digested DNA is sequentially labeled with a marker and blunt-ended to obtain a circularized product; introducing an internal reference circular co-precipitated DNA molecule into the circularized product; and then using a restriction exonuclease Enzyme digestion, then ultrasonic fragmentation, and then use immunomagnetic beads to grab the DNA fragments with the markers from the fragmentation products; use the DNA fragments with the markers to prepare eHi-C 2.0 sequencing library . The present invention can perform eHi-C library construction and sequencing on the number of cells as low as 0.1 million cells.

Description

technical field [0001] The invention relates to the technical field of genome sequencing, in particular to eHi-C 2.0, a genome-wide chromatin conformation technology based on a small number of cells. Background technique [0002] Three-dimensional genomics is an emerging discipline that studies the three-dimensional spatial structure and function of the whole genome (chromatin) [Ruan Y, 2011, Science]; its research scope covers the visual analysis and simulation of the three-dimensional spatial structure of the genome. Genome interaction network research, chromatin dynamic domain, 3D data mining, 3D structure and signaling pathway function analysis, key gene or factor mining and other important research contents [Schmitt A.D, Ren B, 2016, NatureReviews Molecular Cell Biology]. In recent years, the 3D genomics technology based on chromatin conformation capture technology (Chromosome conformation capture) has been greatly developed, which can obtain 3D gene interaction from a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C40B50/06C40B40/06C12N15/10
CPCC12N15/1013C12Q1/6806C12Q1/6869C40B50/06C12Q2521/301C12Q2535/122
Inventor 张玉波孔思远黄其通李琳黄雷白立景彭艳玲
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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