Construction method and application of single-cell methylation sequencing library

A library construction, single-cell technology, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve problems such as affecting library coverage and inability to perform sequencing

Inactive Publication Date: 2018-04-13
HUAZHONG AGRI UNIV
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Problems solved by technology

The operating principle of scBS is to perform bisulfite conversion and fragmentation of the genome first, and then use random primers to perform two-step amplification of small fragments, which can obtain greater coverage and more unbiased methylation positions than scRRBS point information, but the length of small fragments generated by sulfite conversion directly affects the coverage of the library (both too long and too short fragments cannot be sequenced)

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  • Construction method and application of single-cell methylation sequencing library
  • Construction method and application of single-cell methylation sequencing library
  • Construction method and application of single-cell methylation sequencing library

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Embodiment 1

[0025] The operation process of the MB-seq sequencing method adopted by the present invention is as attached figure 1 As shown, the specific operation process is as follows:

[0026] 1. Put the tetrad microspores of corn into 27% mannitol solution, and the intracellular osmotic pressure balance maintains the normal cell shape. Using micro-glass tubes, micro-injectors, and microscopes, isolate single cells or nuclei in 4 μl of pure water. After separation, place the samples on ice until all samples are separated and proceed to the next step or move to a -80°C refrigerator for later use;

[0027] 2. Prepare 8μl cell lysis buffer (15mM Tris-Cl pH 7.4, 0.9% SDS and 0.5μl proteinase K), add the buffer to 4μl sample, incubate at 37°C for 1 hour, after the reaction, you can perform the next step or move to - Standby at 20°C;

[0028]3. Use the Imprint DNA Modification Kit to convert unmethylated cytosine to uracil:

[0029] a) Add 1 μl Balance Solution to the cleaved DNA above, a...

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Abstract

The invention relates to a construction method and application of a single-cell methylation sequencing library. The invention applies a multiple displacement amplification technology to construction of the single-cell methylation sequencing library, and richness and comparison rate of the sequencing library are obviously enhanced. The construction method comprises the following specific steps: firstly producing small segments containing uracil by utilizing an optimized bisulfate conversion kit, then carrying out amplification extension for multiple times by using a label-free random primer, degrading a free single-stranded small segment primer, then denaturalizing a paired double strand by utilizing high temperature, then connecting single-stranded small segments into a single-stranded long fragment, carrying out multiple displacement amplification by utilizing a label-free random primer segment with a terminal 5' closed, obtaining an amplification end product, and finally carrying outlibrary construction based on Tn5 enzyme on a reaction product.

Description

technical field [0001] The invention relates to the field of gene detection. Specifically, it relates to a method for constructing a single-cell genome-wide methylation sequencing library and its application. Background technique [0002] DNA methylation is an important epigenetic mode of genomic DNA, which is catalyzed by DNA methyltransferase (DNAmethyltransferase, DNMT) and S-adenosylmethionine (SAM) Base donor, the process of transferring a methyl group to a specific base. DNA methylation widely exists in bacteria, plants and animals, and participates in various biological processes of organisms (Law J A and Jacobsen S E, Establishing, maintaining and modifying DNA methylation patterns in plants and animals[J].Nat Rev Genet , 2010.11(3): 204-220.). In plants, DNA methylation is specific to species, tissues, organs, cells, and developmental stages, and plays an important regulatory role in plant growth and development, adaptation to environmental conditions, and evolut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869C12Q1/6858
CPCC40B50/06C12N15/1093C12Q1/6858C12Q1/6869C12Q2523/125C12Q2537/137C12Q2537/143
Inventor 李响严建兵陈露
Owner HUAZHONG AGRI UNIV
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