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Method for constructing microscale sample m6A modification detection library under assistance of Tn5 transposase and application thereof

A technology of transposase and sequencing library, which is applied in the field of RNA modification site detection, can solve the problems of inability to meet the needs of trace samples, limitations, and limited applications, and achieves the goal of reducing input, reducing costs, and avoiding contamination of sequencing data. Effect

Active Publication Date: 2021-07-02
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the function of the gene is reflected in the mRNA, and mRNA only accounts for 1-2% of the total RNA, so MeRIP-seq needs to extract mRNA from a large amount of total RNA. In addition, MeRIP-seq obtains RNA samples after antibody immunoprecipitation The initial construction of the sequencing library is limited by the strategy of the ligation method. Therefore, the initial sample of MeRIP-seq usually requires more than 100 micrograms of total RNA as the experimental material, which greatly limits the application of this method to some difficult-to-obtain samples. and application on patient tissue samples
Although the latest research protocol reduces the amount of initial samples to 0.5 micrograms of total RNA by improving the experimental process and strategy, it can also obtain better m6A high-throughput data, but it still cannot meet the needs of micro-samples, especially single cells. needs

Method used

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  • Method for constructing microscale sample m6A modification detection library under assistance of Tn5 transposase and application thereof
  • Method for constructing microscale sample m6A modification detection library under assistance of Tn5 transposase and application thereof
  • Method for constructing microscale sample m6A modification detection library under assistance of Tn5 transposase and application thereof

Examples

Experimental program
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Effect test

example 1

[0068] Example 1 A Tn5 transposase-assisted high-throughput sequencing method for detecting m6A modifications in trace samples

[0069] The primers (Vazyme, cat.no.TD202) needed in this example are as follows (N5-index includes N501-508, N7-index includes N701-712):

[0070]

[0071]

[0072] The total RNA required in this embodiment is 0.06 micrograms, and the process of steps (1)-(7) is as follows figure 1 Shown, the flow process of steps (8)-(16) is as follows figure 2 Shown:

[0073] (1) Using a carbon dioxide incubator, place 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in modified DMEM medium (DMEM; 273 Corning, cat.no.10-017-CV), and add 10% (v / v ) fetal bovine serum (FBS; Gibco), 5% CO 2 , and cultured at 37°C until the number of cells reached the level of 100,000.

[0074] (2) Discard the cell culture medium, wash twice with PBS, digest the cells with trypsin, centrifuge the cell suspension (1000rpm / 5min), discard the supern...

Embodiment 2

[0123] Example 2 A Tn5 transposase-assisted high-throughput sequencing method for detecting m6A modification in trace samples

[0124] The detection sample in this embodiment is 293T cells (2000 cells), that is, the cell lysate is used as the detection sample.

[0125] The primers used in this example are the same as those in Example 1.

[0126] The process of steps (1)-(7) is as follows figure 1 Shown, the flow process of steps (8)-(16) is as follows figure 2 Shown:

[0127] (1) Using a carbon dioxide incubator, 293T cells were placed in modified DMEM medium (DMEM; 273 Corning, cat. no. 10-017-CV) and 10% (v / v) fetal bovine serum (FBS; Gibco) was added.

[0128] (2) Discard the cell culture medium, wash twice with PBS, digest the cells with trypsin, centrifuge the cell suspension (1000rpm / 5min), discard the supernatant, reselect the cells with PBS, and then use flow cytometry Sorter, sort out 2000 cells.

[0129] (3) Add 3 μL of cell lysate to the cells sorted in step ...

Embodiment 3

[0134] Example 3 A Tn5 transposase-assisted high-throughput sequencing method for detecting m6A modification in trace samples

[0135] The detection sample in this embodiment is a drop of blood sample (5 microliters).

[0136] The primers used in this example are the same as those in Example 1.

[0137] The operation flow of this embodiment is as follows Figure 6 Shown:

[0138] (1) Obtain 5 microliters / sample of fingertip peripheral blood from healthy volunteers using a blood collection needle.

[0139] (2) Using erythrocyte lysate (Roche, 11814389001) to remove erythrocytes in peripheral blood to obtain a large number of leukocytes.

[0140] (3) Total RNA was extracted using the classic Trizol (Thermo Fisher Scientific, catalog no. 15596026) method, and operated according to the instructions to obtain total RNA.

[0141] (4) The following m6A modification detection method is the same as that in Example 1, wherein step (11) adopts method two.

[0142] The m6APeak identi...

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Abstract

The invention belongs to the technical field of RNA modification site detection, and particularly relates to a method for constructing a microscale sample m6A modification detection library under the assistance of Tn5 transposase and an application thereof. The m6A modified detection library constructed by the method can greatly reduce the initial input amount of a high-throughput sequencing sample, and the method disclosed by the invention can be used for detecting a trace sample which cannot be applied by the existing MeRIP-seq method, so that the initial total RNA input amount of the high-throughput sequencing sample is reduced to 0.06 microgram.

Description

technical field [0001] The invention belongs to the technical field of RNA modification site detection, and specifically relates to a method for using Tn5 transposase to assist in constructing a micro sample m6A modification detection library and an application thereof. Background technique [0002] It is currently known that there are more than 100 modifications on RNA, including: N6-adenylate methylation (m6A), N1-adenylate methylation (m1A), cytosine hydroxylation (m5C), etc., and m6A It is the most abundant modification among them, affecting the metabolism of RNA, the occurrence and development of tumors, and the growth and development of cells. [0003] Currently known m6A detection methods mainly fall into two categories: one is mass spectrometry, which can detect the overall level of m6A modification in a sample, but it is impossible to know the specific modification position of m6A modification and which genes are modified. The other is high-throughput sequencing me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06C12Q1/6869
CPCC12Q1/6806C40B50/06C12Q1/6869C12Q2525/191C12Q2521/50C12Q2523/308C12Q2535/122
Inventor 骆观正陈涛李言马冬曌张璋奚剑飞
Owner SUN YAT SEN UNIV
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