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A method to detect nucleosome arrangement on the genome at the single-cell level

A genome and nucleosome technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as difficulties, heterogeneity of gene expression in cell populations, and interaction of transcription factors, so as to reduce losses, Effects of reducing DNA purification times and improving construction efficiency

Active Publication Date: 2021-01-15
SHANGHAI FIRST MATERNITY & INFANT HOSPITAL +1
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  • Description
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AI Technical Summary

Problems solved by technology

[0006] Recent studies have shown that the position of nucleosomes affects the interaction of transcription factors and affects the expression status and expression levels of genes in different cells, resulting in the heterogeneity of gene expression in cell populations
Traditional MNase-seq requires millions of cells to perform a test, and the result obtained is the average state of these cells, and the differences in nucleosome arrangement between different cells are ignored, and these differences are what lead to cell heterogeneity In addition, in many cases, especially for cells in the body, it is very difficult to obtain the millions of cells required by traditional MNase-seq, so it is very difficult to develop a method suitable for a small number of cells or even single cells MNase-seq technology is crucial to solving these problems

Method used

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  • A method to detect nucleosome arrangement on the genome at the single-cell level
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  • A method to detect nucleosome arrangement on the genome at the single-cell level

Examples

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Embodiment 1

[0030] Example 1: MNase-seq detection of a small amount or a single mouse embryonic stem cell

[0031] 1. Purpose of the experiment:

[0032] The method of the invention is used to detect the nucleosome arrangement of a single or a small number of embryonic stem cells, and construct a DNA library that can be used for detection on an illumina machine.

[0033] 2. Experimental method:

[0034] 1. Cell acquisition

[0035] 1) The mouse embryonic stem cell line R1 was purchased from American Type Culture Collection (ATCC), and cultured and passaged in the laboratory.

[0036] 2) Pick a mouse embryonic stem cell clone with a mouth pipette, and wash it once in Duchenne's phosphate buffered solution (DPBS).

[0037] 3) Place the clone in trypsin at 37°C for 5 minutes.

[0038] 4) Blow and aspirate the clone repeatedly with a mouth pipette with a diameter of about 10 μm until all the cells in the clone are scattered into single cells.

[0039] 5) Wash the cells three times in pho...

Embodiment 2

[0082] Example 2: MNase-seq detection of prokaryotic

[0083] 1. Purpose of the experiment:

[0084] The method of the invention is used to detect the arrangement of nucleosomes in prokaryotes at the early stage of fertilization, and to construct a DNA library that can be used for detection on an illumina machine. In this way, it is tested whether the method of the present invention is applicable to the nucleosome arrangement detection of the haploid genome.

[0085] 2. Experimental method:

[0086] 1. Acquisition of pronuclei

[0087] 1) The SPF grade C57BL / 6 mice used in the experiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and were bred in the Experimental Animal Center of Tongji University.

[0088] 2) Take 8-10 week-old female mice, intraperitoneally inject 5U of pregnant horse serum gonadotropin (PMSG), and 48 hours later intraperitoneally inject 6U of human chorionic gonadotropin (hCG). The hormone-injected female mice we...

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Abstract

The invention relates to a method for detecting nucleosome arrangement on a genome at single cell level. The method includes the steps: (1) lysing cells and treating DNA (deoxyribonucleic acid) fragments on the genome by micro-sphere nuclease; (2) treating a product after enzyme digestion in the step (1) by protease and removing histone wound on the DNA fragments; (3) performing sequencing library construction on the DNA fragments obtained in the step (2) and performing high-throughput sequencing; (4) performing comparative analysis on high-throughput sequencing results, and assessing the quality of the sequencing results including coverage on the genome, fragment length distribution and nucleosome arrangement at a transcriptional start site and a CTCF binding site. Loss is decreased by reducing DNA purification frequency as far as possible, library construction efficiency is improved, and further minimum cells and even single cell can be used for detecting cell nucleosome arrangement at the whole genome level.

Description

technical field [0001] The invention relates to a method for detecting the arrangement of nucleosomes on the genome, especially a method for detecting the arrangement of nucleosomes in single cells or even haploid cells. Background technique [0002] Cell is the basic unit of organism function, and the coordinated work of a large number of cells ensures the normal and orderly progress of life activities. However, in some diseases, such as cancer, the excessive proliferation of a single cell can cause the disorder of the whole organ function. So far, most genome-level detections have been carried out on a large number of cells, and the data obtained are the average of these samples, which makes it difficult to explain the differences between cells. Similarly, using traditional methods for some cells Sequencing small samples, such as preimplantation embryos, is difficult. The realization of single-cell sequencing technology has opened up a new field of genomics research, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122
Inventor 高绍荣高亚威刘晓雨陈川陈嘉瑜陈珺
Owner SHANGHAI FIRST MATERNITY & INFANT HOSPITAL
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