Library establishing method for unicellular RRBS sequencing and application thereof

An established method and single-cell technology, applied in the field of molecular biology, can solve the problems of cross-contamination between samples, low input sample volume, loss of useful information, etc.

Inactive Publication Date: 2017-12-15
上海美吉医学检验有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0008] The existing restriction endonuclease-bisulfite sequencing method requires complex experimental procedures: MspI digestion, electrophoresis, and purification steps to enrich the promoter and CpG island regions, followed by end repair, A addition, adapter ligation, Bisulfite conversion, purification, PCR amplification, PAGE gel fragment selection and other steps to construct a sequencing library will take about 5-6 days
Moreover, after adding adapters and perfor

Method used

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  • Library establishing method for unicellular RRBS sequencing and application thereof
  • Library establishing method for unicellular RRBS sequencing and application thereof
  • Library establishing method for unicellular RRBS sequencing and application thereof

Examples

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Embodiment 1

[0126] Single-cell samples can be lysed or multi-cell samples can be extracted to obtain sample genomic DNA. Sample genomic DNA that has been obtained by lysis of a single cell sample or extracted from a multicellular sample by others using techniques well known in the art can also be used.

[0127] As an example: the sample genomic DNA in the examples of this application can be obtained from the following steps 1 and 2:

[0128] Step 1. Single cell isolation.

[0129] Single or multiple cells can be obtained using cell separation methods such as mouth aspiration, laser microscopic capture, limiting dilution, and flow cytometry. The buffer containing cells is preferably 1×PBS, and can also be an isotonic solution of cells such as physiological saline and 0.278mol / L sucrose, with a volume of 1-4 μl.

[0130] Step 2. Cell lysis.

[0131] 2a) according to the system of table 1 to establish cracking system:

[0132] Table 1

[0133] Element

Volume (ul)

Final co...

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Abstract

The invention relates to the technical field of molecular biologics and specifically discloses a library establishing method for unicellular RRBS sequencing and an application thereof. The library establishing method for unicellular RRBS sequencing, disclosed by the invention, comprises the following steps: (1) performing digestion, terminal filling, A and joint adding and bisulfate conversion on genomic DNA of a sample in turn; (2) performing linear amplification on the genomic DNA converted in the step (1); (3) performing index amplification on the amplicon subjected to linear amplification in the step (2), wherein the amplicon of the index amplification is used for sequencing the library. The DNA required by the sequencing according to the method is low in initial mass; methylation sequencing can be performed on the genome of a single cell; the method contains an effective library segment control step; the segment of the end product is controlled according to the linear amplification step; complex operation is avoided while the coverage rate is increased.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a method for establishing a library suitable for representative bisulfite sequencing (Reduced Representation Bisulfite Sequencing, RRBS) sequencing of single cells and its application. Background technique [0002] DNA methylation refers to the covalent modification of the fifth carbon atom of cytosine (C) on DNA into 5' methylcytosine (5mC). DNA methylation is one of the earliest discovered epigenetic modification pathways, which can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. It has a variety of important biological functions, including X chromosome inactivation, transposon silencing, gene imprinting, etc. The vast majority of DNA methylation modifications occur at CpG sites. In mammals, CpG exists in two forms: one is dispersed in the DNA sequence; the o...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/68
CPCC40B50/06C12Q1/6858C12Q1/6869C12Q2535/122C12Q2523/125C12Q2525/191
Inventor 李静陈昌岳王芳张祥林胡秋萍董东郑冠涛段彪
Owner 上海美吉医学检验有限公司
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