New method for capturing chromatin nucleosome vacancy district at high-throughput complete genome level and use therefor

A genome-wide and chromatin technology, applied in the field of medical molecular biology, can solve problems such as low accuracy

Inactive Publication Date: 2012-09-26
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Description
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Problems solved by technology

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  • New method for capturing chromatin nucleosome vacancy district at high-throughput complete genome level and use therefor
  • New method for capturing chromatin nucleosome vacancy district at high-throughput complete genome level and use therefor
  • New method for capturing chromatin nucleosome vacancy district at high-throughput complete genome level and use therefor

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Embodiment 1

[0026] Step 1. Lyse cells and extract nuclei

[0027] HeLa S3 cells were cultured to reach 80% confluency and replaced with serum-free medium. After 24 hours of serum starvation [15, 16], the culture medium was discarded. Wash the cells twice with ice-cold PBS, then add 1ml of PBS, scrape the cells on ice with a cell scraper, centrifuge at 3000rpm for 10 minutes at 4°C, and collect the cells. The cells were suspended with PBS buffer, and the cells were counted. 10 7 Aliquot / tube and centrifuge again to collect cells. Add nuclei isolation buffer (10mM Tris buffer, pH 7.4, 10mM NaCl, 5mM MgCl 2 , 1mM PMSF, 0.2% NP40), mix gently to make the final cell concentration 5×10 6 / ml, ice bath for 10 minutes. Centrifuge at 3000 rpm for 10 minutes at 4°C to collect cell nuclei. Nuclei were washed with NP40-free buffer (10 mM Tris buffer, pH 7.4, 10 mM NaCl, 5 mM MgCl 2 , 1 mM PMSF), washed away excess NP40, and centrifuged at 3000 rpm for 10 minutes at 4°C.

[0028] Step 2. Neutr...

Embodiment 2

[0074] Step 1. High-throughput sequencing of NFR library

[0075] 1. Preparation of sequencing samples

[0076] The NFR library preparation method is the same as the above step 1 to step 8, extract HeLaS3 nucleus; neutral formaldehyde fixes the nucleus; DNase I makes nick; nick DNA translation biotin incorporation; S1 nuclease cut nick DNA; phenol / chloroform extraction, ethanol precipitation; DNA A was added to the end of the fragment, and a linker was added; biotin-DNA was picked up by magnetic beads; NFR library was amplified; DNA fragments were recovered from the gel; The samples were sent to BGI and Illumina's Solexa Genome Analyzer platform for high-throughput sequencing.

[0077] 2. The distribution of Unique mapped reads in coding regions and intron regions. Use the known information of UCSC to annotate the Sequencing data, calculate the proportion of exons, introns and conserved non-coding regions in the entire Sequencing data, and compare it with the whole genome da...

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Abstract

The invention relates to a new method for high-efficiently capturing a chromatin nucleosome vacancy district at a whole-cell level. The new method can locate the chromatin nucleosome vacancy district more accurately and sensitively in the range of a complete genome. The method for capturing the chromatin nucleosome vacancy district at a high-throughput complete genome level can be used to search the chromatin nucleosome vacancy district of the transcriptional regulation of eukaryotic cells, and is an effective means for functional genomics research.

Description

technical field [0001] The invention belongs to the field of medical molecular biology and relates to a new method for capturing chromatin nucleosome vacant regions at the level of high-throughput whole genome. Background technique [0002] The main content of research in the post-genome era is the regulatory mechanism of genes at the genome level, in which nucleosome positioning, its chemical composition, and the modification of its components are important research contents [1-6]. The basic structural unit of chromatin is the nucleosome. It is the nucleosome that compresses the length of the DNA linear structure by about 10,000 times. While compressing the genome, the nucleosome structure also limits the binding between transcription factors and DNA. Two molecules of histones H2A, H2B, H3 and H4 constitute the core histone of the octamer. A DNA molecule with a length of about 146 bp coils 1.65 turns on the histone octamer to form the core particle of the nucleosome, and e...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08C12Q1/68
Inventor 张玉祥李振华王雅梅周萍
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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