Efficient whole-genome chromosome conformation capture technology (eHi-C)

A chromatin and ehi-c technology, which is applied in the direction of recombinant DNA technology, combinatorial chemistry, microbial measurement/testing, etc., can solve the problems of increased cost of cell culture, increased experimental error, and low resolution of interaction, etc. Effects of required amount of cells, saving reagents, and simplifying steps

Active Publication Date: 2017-04-19
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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Problems solved by technology

The amount of cells required by the standard test technique is relatively large, and it takes time to collect enough cells; the number of experimental materials required is large, which leads to

Method used

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  • Efficient whole-genome chromosome conformation capture technology (eHi-C)
  • Efficient whole-genome chromosome conformation capture technology (eHi-C)
  • Efficient whole-genome chromosome conformation capture technology (eHi-C)

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1. Preparation of cell eHi-C sequencing library and its application in sequencing (5-standard)

[0069] figure 1 It is a flowchart of main steps of the present invention.

[0070] (1) Lysis cells to prepare chromatin

[0071] 1. Cell collection and cross-linking fixation

[0072] 1. When the cell confluence rate in the culture dish (10CM) reaches 70-80%, the C2C12 cells ( CRL-1772 TM ) counts about 2million, such as image 3 , a, cell state (magnification 100×); b, result of automatic cell counter), the volume of the culture solution was detected by pipetting;

[0073] 2. Add 37% formaldehyde aqueous solution (280 ul) by 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);

[0074] 3. Add 2.5M glycine aqueous solution (0.89ml, so that the final concentration is 0.2M) to quench the formaldehyde, put it back into the rotator and sha...

Embodiment 2

[0150] Example 2, other methods of cell eHi-C sequencing

[0151] 1. Method ( figure 2 shown):

[0152] 1. 1-Option group: the method is basically the same as in Example 1, the only difference is (2), to obtain the 1-Option group eHi-C sequencing library:

[0153] Use 500ul of 1×NEBuffer 2 (NEB; #B7002S) to wash the chromatin aggregate obtained in (1) above, centrifuge at 20,000g for 15min at 4°C, and remove the supernatant. repeat. Break up the clumps again with a pipette tip, add 38ul 1×NEBuffer 2, resuspend and mix as completely as possible, and obtain the resuspended chromatin.

[0154] Add 3.8 ul of 1% SDS aqueous solution to the resuspended chromatin, so that the final concentration of SDS is 0.1%, mix well, and incubate at 65°C for 10 minutes to remove proteins that are not directly cross-linked with DNA; after incubation Immediately put the centrifuge tube on ice, add 4.4ul volume percentage of 10% Triton X-100 to quench SDS, mix carefully to avoid air bubbles; get...

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Abstract

The invention discloses an efficient whole-genome chromosome conformation capture technology (eHi-C). The invention provides an eHi-C sequencing technology for cells. The eHi-C sequencing technology comprises the following steps: 1) subjecting the cells to disintegration so as to obtain chromatin; 2) subjecting the chromatin to enzyme digestion, DNA end labeling, cyclization connection of a blunt end and DNA purification so as to obtain purified circular DNA; 3) introducing a circular co-precipitation DNA molecule used as internal reference; 4) carrying out ultrasonic breaking; 5) capturing all the labeled DNA fragments with immunomagnetic beads; and 6) preparing eHi-C sequencing library from the labeled DNA fragments. The technology provided by invention is low in the amount of needed cells, can easily acquire an experiment material, reduces time in acquisition of the material, greatly lowers cell culture cost and cost for reagent consumables and decreases test errors.

Description

technical field [0001] The invention relates to the technical field of genome sequencing, in particular to eHi-C, an efficient genome-wide chromatin conformation technology. Background technique [0002] Three-dimensional (3D) genomics is a discipline that studies the three-dimensional spatial structure and function of the genome. Specifically, it refers to the study of genome sequence, gene structure and its regulatory elements, the three-dimensional spatial structure of genome sequence in the nucleus, and its function in biological processes such as gene transcription, replication, repair, and regulation. The related sequencing technologies of 3D genome mainly include Hi-C (High-throughput / resolution chromosome conformationcapture) developed by Job Dekker and ChIA-PET (Chromatin Interaction Analysis with Paired-End Tag Sequencing) technology developed by Professor Ruan Yijun (Li Guoliang, Ruan Yijun, 2014, Science Bulletin). [0003] Hi-C technology is based on high-thro...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C12Q1/68
Inventor 张玉波孔思远黄其通黄雷王秋雁白立景张高林钟学优
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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