Unicell simplified representative bisulfite sequencing method and kit

A bisulfite and single-cell technology, applied in biochemical equipment and methods, chemical libraries, combinatorial chemistry, etc., can solve the problem of low coverage of CpG islands

Pending Publication Date: 2016-04-20
ANNOROAD GENE TECH BEIJING +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the experimental analysis results obtained by this method have low coverage of CpG islands

Method used

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  • Unicell simplified representative bisulfite sequencing method and kit
  • Unicell simplified representative bisulfite sequencing method and kit
  • Unicell simplified representative bisulfite sequencing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Using mouse oocytes as materials to construct scRRBS (single-cell simplified representative bisulfite) sequencing library

[0072] (1) lysed cells

[0073] Isolate a single mouse oocyte and place it in a 1.5mL sampling tube with a volume of about 1μL; add 1μL of ddH to the negative control tube 2 O. Without transferring the tube, directly add 5 μL LysisBuffer, 1 μL Protease, and 1 μL λDNA to the cells in a total of 7 μL, the total volume is 8 μL, and wrap the parafilm to seal. Submerge the entire small tube in a water bath at 50°C for 3 hours, and then inactivate Protease at 70°C for 15 minutes. A mixed solution containing mouse oocyte genomic DNA was obtained.

[0074] (2) MspI digestion

[0075] Digest mouse oocyte genomic DNA according to the following system, wherein the volume of the mixture (mouse oocyte gDNA) obtained from step (1) pretreatment is 8 μL (already containing λDNA),

[0076]

[0077] Submerge the entire vial in a water bath at 37°C ...

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Abstract

The invention relates to a unicell simplified representative bisulfite sequencing library construction method which comprises the steps of unicell pyrolysis, MspI enzyme digestion, tail end repair, 3'- terminal A addition, methylated linker addition, bisulfite treatment and PCR (polymerase chain reaction) amplification. The invention also relates to a unicell simplified representative bisulfite sequencing library construction kit, and a detection method and detection kit thereof, belonging to the field of gene sequencing. By using the unicells as the research object, the unicell simplified representative bisulfite sequencing library construction method for sequencing can be used for researching cell heterogeneity on the unicell level.

Description

technical field [0001] The invention relates to a construction method of a single-cell simplified representative bisulfite sequencing library, a library construction kit, a detection method and a detection kit, belonging to the field of gene sequencing. Background technique [0002] DNA methylation is one of the earliest epigenetic modification methods discovered in eukaryotes. Methylation in eukaryotes mainly occurs at cytosine, that is, under the action of DNA methyltransferases (DNMTs), CpG dinuclear Cytosine (C) at the 5'-end of the nucleotide is converted to 5'-methylcytosine (5mC). The main function of methylation is to regulate gene expression. In mammals, DNA methylation is relatively stable in differentiated cells; by contrast, genome-wide demethylation and DNA methylation re-establishment occur during early embryonic development and primordial germ cell development (Remethylation) process. [0003] Whole-genome sulfite sequencing can comprehensively analyze DNA ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C40B50/06C40B40/06
CPCC12Q1/6869C12Q1/6876C12Q2600/154C40B40/06C40B50/06C12Q2525/191C12Q2531/113C12Q2521/501
Inventor 柳青王晓雯洪燕姜涛赵红梅张广思玄兆伶李大为梁峻彬陈重建
Owner ANNOROAD GENE TECH BEIJING
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