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Method for constructing multiple single-cell simplified representative methylation library based on Illumina sequencing platform

A sequencing platform and construction method technology, applied in the field of molecular biology, can solve problems such as DNA loss and DNA fragmentation, and achieve the effect of improving coverage and saving construction time

Pending Publication Date: 2020-07-10
杭州圣庭医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The harsh conditions of sodium bisulfite treatment can lead to random fragmentation of DNA, which can lead to substantial loss of DNA if the library DNA is equipped with sequencing adapters prior to bisulfite treatment

Method used

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  • Method for constructing multiple single-cell simplified representative methylation library based on Illumina sequencing platform
  • Method for constructing multiple single-cell simplified representative methylation library based on Illumina sequencing platform
  • Method for constructing multiple single-cell simplified representative methylation library based on Illumina sequencing platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 single cell lysis

[0077] 1. Dispense 5 μl of 0.1x CutSmart Buffer to each well in a 96-well PCR plate. The CutSmart buffer, that is, the cell lysate component is 10 mM Tris-HCl (PH=7.0), 10 mM EDTA, 10 mM NaCl and 0.1% (wt / vol) SDS.

[0078] 2. Sorting one single cell per well in a 96-well plate. If the sorted cells are not ready for making single-cell reduced representative methylation libraries, the 96-well plate can be sealed with a PCR Seal "F" membrane (BIORAD, CAT#MSF1001) and the It was stored in a -80°C freezer until further processing.

[0079] 3. Prepare the cell lysis buffer mix according to the table below.

[0080]

[0081] Note: Prepare an additional 20% more master mix to compensate for loss of reagents during pipetting.

[0082] 4. Distribute 19 μl of the above buffer solution to each well of 12 wells in row A of a 96-well PCR plate, seal the 96-well plate and perform short centrifugation, and then use a 12-channel pipette to transfer ...

Embodiment 2

[0090] Embodiment 2 Restriction endonuclease digestion

[0091] 1. Prepare the MspI digestion reaction mixture as follows:

[0092]

[0093] Note: Another methylation-insensitive restriction enzyme can be added to increase CpG island coverage. Make sure different restriction enzymes are compatible in the same CutSmart buffer.

[0094] 2. Dispense 19 μl of the Msp I digestion reaction mixture into each of the 12 wells in row C of a 96-well PCR plate.

[0095] 3. Carefully remove the cover film of the 96-well sample plate to avoid cross-contamination of the sample, and then use a 12-channel pipette to transfer 2 μl of the digestion mixture to each sample well. Briefly centrifuge the 96-well plate.

[0096] 4. Mix the reaction by shaking gently and centrifuge briefly.

[0097] 5. Put the sample plate into the thermal cycler, and the temperature of the thermal cover is preset to 80°C. Follow the procedure below to incubate the reaction:

[0098] program Temper...

Embodiment 3

[0101] Example 3 End Repair

[0102] 1. Prepare the end repair system according to the following formula:

[0103]

[0104] 2. Distribute 19 μl of the above reaction system to each well of 12 wells in row E of a 96-well PCR plate.

[0105] 3. Carefully remove the cover membrane of the 96-well sample plate to avoid cross-contamination of samples, and then use a 12-channel pipette to transfer 2 μl of end repair mixture to each sample well. Briefly centrifuge the 96-well plate.

[0106]4. Mix the reaction by shaking gently and centrifuge briefly.

[0107] 5. Put the sample plate into the thermal cycler, and the temperature of the thermal cover is preset to 80°C. Follow the procedure below to incubate the reaction:

[0108] program Temperature(°C) Duration (Minutes) 1 30 10 2 37 10 3 70 5 4 4 hold

[0109] 6. After the incubation, centrifuge the sample plate at 3000 rpm for 30 seconds.

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Abstract

The invention provides a method for constructing a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform. The method comprises the following steps: (1) single-cell lysis or trace DNA extraction and purification; (2) digestion with restriction endonuclease Msp I; (3) repairing the tail end and adding A to the 3'end; (4) connecting methylated joints with labels; (5) converting the hydrosulfite into non-methylated cytosine; (6) carrying out PCR enrichment on the methylation library; and (7) carrying out mixing and Illumina platform sequencingon methylated libraries with similar sizes. The method not only solves the problem of insufficient unicellular methylation sequencing initial quantity, but also can perform large-batch operation treatment by introducing a double-label sequence into each unicellular sample and is easy to realize an automatic process. The method not only reduces the methylated library construction cost of each sample, but also shortens the library construction time.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform. Background technique [0002] DNA methylation is a form of chemical modification of DNA that can alter genetic expression without altering the DNA sequence. DNA methylation refers to the covalent bonding of a methyl group at the 5' carbon position of cytosine of a genomic CpG dinucleotide under the action of DNA methyltransferase. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction between DNA and proteins, thereby controlling gene expression. [0003] In mammals, CpG exists in two forms: one is dispersed in the DNA sequence; the other is highly aggregated, which is called CpG island (CpG island). In normal tissues, 70% to 90% of the scattered ...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12Q1/6869
CPCC40B50/06C40B40/06C12Q1/6869C12Q2531/113C12Q2535/122C12Q2525/191C12Q2523/125
Inventor 谷红仓许佩松王云飞车仙荣
Owner 杭州圣庭医疗科技有限公司
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