Hi-C high-throughput sequencing and database building method for eukaryote DNA

A eukaryotic, high-throughput technology, applied in the field of biochemistry, can solve problems such as limited applications, and achieve the effects of wide applicability, improved cyclization efficiency, and simple operation procedures

Active Publication Date: 2016-08-10
BIOMARKER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The above defects of the existing Hi-C high-throughput sequencing limit the application of this technology in promoting functional genomics research, so it is necessary to improve this technology to provide a new Hi-C with high data rate and wide applicability. High-throughput sequencing library construction method

Method used

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  • Hi-C high-throughput sequencing and database building method for eukaryote DNA
  • Hi-C high-throughput sequencing and database building method for eukaryote DNA
  • Hi-C high-throughput sequencing and database building method for eukaryote DNA

Examples

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Embodiment 1

[0044] Example 1 The high-throughput sequencing library construction method suitable for rice Hi-C of the present invention

[0045] 1. Collect 1g of fresh leaves (to ensure the integrity of chromosome interaction), and put them on ice.

[0046] 2. Place the collected fresh tissue on a 50ml centrifuge tube, add 15ml of pre-cooled 4°C NIB buffer (add 15μl of mercaptoethanol before use) and 1ml of 36% formaldehyde solution, and incubate at 25°C for 40min. Just make sure the sample sinks to the bottom of the tube to ensure sufficient cross-linking. Add 2M glycine to make the final concentration 0.1M, and react under vacuum condition for 5min, all the samples are washed several times with ultrapure water, and the samples are ground into powder with liquid nitrogen.

[0047] Transfer 3.1 g of tissue samples to 10 ml of pre-cooled 4°C NIB buffer (in which the concentration of protease inhibitors is 0.1 mM), shake slowly on ice for 15 minutes, and filter the suspension twice with a ...

Embodiment 2

[0070] Example 2 The present invention is suitable for human blood Hi-C high-throughput sequencing library construction method and its comparison with the library construction method not of the present invention

[0071] 1. Experimental steps and results of the Hi-C library construction method not of the present invention:

[0072] 1. Collect white blood cells in 1ml of fresh blood (to ensure the integrity of chromosome interaction), and place on ice.

[0073]2. Place the collected white blood cells in a 15ml centrifuge tube and resuspend with 12ml of freshly prepared PBS. Add 686 μl of 37% formaldehyde (final concentration 2%), and mix by inverting at room temperature for 10 min. Add 1.8ml of 1M glycine (final concentration 0.125M), and mix for 2-3min. Centrifuge at 3000rpm for 20min to collect white blood cells; resuspend the pellet once in pre-cooled 4°C PBS, and centrifuge to remove the supernatant. Resuspend leukocytes in 5ml of pre-cooled 4°C lysis buffer (with protea...

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Abstract

The invention relates to a Hi-C high-throughput sequencing and database building method for eukaryote DNA. According to the DNA-cell-nucleuse intramolecular cyclization step, end-repaired DNA and a cyclizing system are mixed, incubated for 2 h-4 h at the temperature of 4 DEG C to 6 DEG C and then incubated for 4 h-6 h at the temperature of 16 DEG C to 22 DEG C, and materials obtained after intramolecular cyclization are obtained, wherein the ingredients of the cyclizing system comprise T4 ligase damping liquid, BSA, T4 DNA ligase and at least one polyethylene glycol selected from PEG 4000 and PEG 6000. By means of the Hi-C high-throughput sequencing and database building method, the character cyclizing efficiency of DNA can be effectively improved, and the effective data rate obviously higher than that of a traditional method is obtained.

Description

technical field [0001] The invention belongs to the field of biochemistry, and specifically relates to a method for building a Hi-C high-throughput sequencing library of eukaryotic DNA. Background technique [0002] Chromosome Conformation Capture (3C) technology is a technology for studying chromosome-protein interaction and chromosome conformation. Under the guidance of second-generation sequencing technology, several methods have been developed. The first of these methods is Hi-C technology published in 2009. In Hi-C technology, the entire nucleus is taken as the research object, using high-throughput sequencing technology, combined with bioinformatics methods, to study the spatial position relationship of the entire chromatin DNA within the genome; Interaction patterns are captured to obtain high-resolution 3D structural information of chromatin. Hi-C technology is widely used and runs through the frontier and hot areas of life science research. The existing Hi-C tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/68
CPCC12N15/1093C12Q1/6869C12Q2521/537C12Q2521/301C12Q2521/501C12Q2535/122
Inventor 郑洪坤刘慧宋军郭艳
Owner BIOMARKER TECH
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