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A single-cell methylation sequencing technology and its application

A single-cell, methylation technology, applied in the field of single-cell methylation sequencing technology, can solve the problems of low sequencing data mapping rate, data waste, time-consuming and labor-intensive, etc., and reduce the probability of combining random primers and adapter sequences , high Mapping rate, and short manual operation time

Active Publication Date: 2022-02-01
CAPITALBIO CORP
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Problems solved by technology

However, this method is extremely complicated, and the main steps include cell lysis, BS transformation, pre-amplification with biotin-labeled pre-amplification primers, magnetic bead capture and recovery of the pre-amplified first strand, ExoI primer digestion, and Oligo2 primer pre-amplification And subsequent PCR amplification and indexing
This process is very time-consuming and complicated. During each cycle of the amplification process, Klenow enzyme needs to be newly added. The steps are complicated, time-consuming and labor-intensive.
Responding to multiple opening operations, it is easy to cause pollution, bring great difficulties to subsequent analysis, and cause data waste. The mapping rate of sequencing data is low. The mapping rate reported in the literature is only about 10-30%, resulting in a large amount of data. waste

Method used

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  • A single-cell methylation sequencing technology and its application
  • A single-cell methylation sequencing technology and its application
  • A single-cell methylation sequencing technology and its application

Examples

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Embodiment 1

[0040] Example 1. Establishment of single-cell methylation sequencing method

[0041] For the process of building a database, see figure 1 . In this embodiment, A549 cells are used as an example for operation.

[0042] 1. Single cell picking

[0043] 1. Take 2.5 μL single cell lysate buffer (BufferRLT Plus, QIAGEN, catalog number: 1053393), put it in a 0.2 mL centrifuge tube, centrifuge briefly (≤600 g, ~5 sec), and set aside.

[0044] 2. Pick a single A549 cell, place it in the lysate from step 1, and lyse it at room temperature for 10 minutes.

[0045] 2. Bisulfite conversion

[0046] Any bisulfite conversion kit can be used for bisulfite conversion.

[0047] In this example, the EZ DNAMethylation-Lightning kit (ZYMO research) was used for bisulfite conversion. The specific steps were as follows:

[0048] (1) Incorporate a certain amount of Lambda DNA into each cell, add H 2 O to a final volume of 10 μL.

[0049] (2) Add 600μL M-Binding Buffer to Zymo-Spin TM IC Co...

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Abstract

The invention discloses a single-cell methylation sequencing technology and its application. The present invention has the following advantages: (1) simple operation, short manual operation time and less pollution. (2) According to the characteristics of the genome sequence after bisulfate treatment, design pre-amplification primers. The 3' end of the preamplification primer is H base. The H base is not complementary to C, and does not affect the combination of preamplification primers and bisulfite-converted sequences, but it can reduce the probability of random primers and linker sequences binding; (3) has a higher Mapping rate and is effective The utilization rate of sequencing data is improved, which is 20%-30% higher than the current scRRBS (average 25%) and scBS (average 10-30%). The invention has great significance for the study of single cell DNA methylation.

Description

technical field [0001] The invention relates to a single-cell methylation sequencing technology and its application. Background technique [0002] Recent studies have shown that DNA methylation plays a key regulatory role in many biological processes, such as the regulation of gene expression, the activity of transposons, the inactivation of X chromosome, and the maintenance of genomic imprinting. Genomic DNA methylation is tissue-specific and is closely related to development and aging. Abnormal DNA methylation usually occurs in the genome of many diseases and even cancer cells. At present, the study of genome-wide methylation at the single-cell level has become a research hotspot, which is of great significance for the discovery of epigenetic information in highly heterogeneous cells. Large-scale single-cell epigenetic analysis can be used in embryonic development, Complicated diseases such as the regulation mechanism of cancer occurrence, cell heterogeneity, early detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6858
CPCC12Q1/6858C40B50/06C12Q2535/122C12Q2525/191C12Q2523/125C12Q2531/113
Inventor 王辉郭弘妍邓莉莉邢婉丽程京
Owner CAPITALBIO CORP
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