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RNA link tag-based low-sample-size m6A high-throughput sequencing method

A sample size, high-throughput technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of low modification content, achieve the effect of simplifying experimental operations, reducing experimental costs, and reducing consumption

Pending Publication Date: 2021-12-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since MeRIP-seq etc. utilize m 6 A high-throughput sequencing method for specific recognition of antibodies is affected by the efficiency of antibody immunoprecipitation and m 6 Influenced by the fact that the A modification content itself is low, it is difficult to achieve m for a single clinical peripheral blood sample (2-4mL whole blood sample) 6 A-seq

Method used

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  • RNA link tag-based low-sample-size m6A high-throughput sequencing method
  • RNA link tag-based low-sample-size m6A high-throughput sequencing method
  • RNA link tag-based low-sample-size m6A high-throughput sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Adenylation of 3'-linker:

[0050] (1) Mix the 3'-linker (see the table for the sequence) with ATP, T4 PNK Buffer and T4 PNK enzyme and then react. The specific treatment method is: dissolve the ordered 3'-linker in enzyme-free water to a final concentration of 100 μM, Then 5 μL was taken out and added to the following reaction system (Table 3) to react at 37°C for 1 hour, followed by inactivation at 65°C for 20 minutes.

[0051] Table 3 Phosphorylation system (50μL)

[0052] 3'linker (100μM) 5μL 10mM ATP 3μL 10×T4 PNK Buffer 5μL 10U / μL T4 PNK enzyme 2μL Enzyme-free water to 50μL

[0053] The reaction system was purified using the Oligo Clean & Concentrator (OCC, Zymo) purification kit, and then eluted with 10 μL of enzyme-free water to obtain the phosphorylated 3’-linker.

[0054] (2) The phosphorylated 3'-linker obtained in step (1) was reacted with Mth RNA Ligase, 5'DNA adenylation reaction buffer, and ATP in the followin...

Embodiment 2

[0059] Adenylation reaction verification:

[0060] Take an equal amount of the adenylated sample and the 3'-linker that has not been adenylated, add loading buffer and perform 20% neutral polyacrylamide gel electrophoresis.

[0061] Analysis of results:

[0062] figure 2 It is the polyacrylamide gel electrophoresis picture of the 3'-linker after adenylation treatment in the present invention. It can be seen from the figure that the 3'-linker has been successfully adenylated, but there are still a small amount of substrates that have not been adenylated. Since the unsuccessfully adenylated 3'-linker has no ligation activity and the 3'-linker used in the reaction is in excess, it will not affect the subsequent experiments.

Embodiment 3

[0064] 1. Library construction:

[0065] Use Trizol reagent to extract RNA in samples (cells, tissues, blood samples, etc.), and then interrupt different RNA samples according to the manual of magnesium ion chemical interrupting reagent (NEB, E6150S), and use RNAClean&Concentrator to interrupt the RNA (RCC) purification kit (Zymo) for purification, and then eluted with 7 μL of enzyme-free water. The eluted RNA was added to the following system (Table 5) for PNK treatment at 37°C for 1 h, so that it could react with the adenylated 3'linker.

[0066] Table 5 PNK treatment system (10μL)

[0067] Fragmented and purified RNA 7μL RibiLock RNase Inhibitor (40U / μL) 1μL 10×T4 PNK Buffer 1μL 10U / μL T4 PNK enzyme 1μL

[0068] As shown in Table 6 below, add 3'linker, T4 Ligase 2 (truncatedKQ) and other reagents required for the ligation reaction directly into the PNK reaction system, pipette up and down with a pipette gun to mix well, react at 25°C f...

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Abstract

The invention discloses a RNA link tag-based low-sample-size m6A high-throughput sequencing method, and belongs to the field of high-throughput sequencing of RNA. The method disclosed by the invention comprises the following steps: carrying out phosphorylation treatment and adenylation treatment on a 3'-linker containing different barcode tag sequences; the adenylation-treated 3'-linker is linked to interrupted RNA samples of different sources; after the samples are mixed, an input control sample is reserved for RNA-seq, m6A antibody immunoprecipitation is conducted on the remaining mixed samples to obtain IP samples and the IP samples are used for m6A-seq, and finally a next-generation sequencing library is obtained and sequencing is performed; sequencing data is split and analyzed according to a barcode sequence to obtain RNA-seq and m6A-seq information of an initial single sample. The RNA link tag-based low-sample-size m6A high-throughput sequencing method can implement m6A antibody immunoprecipitation and library-building sequencing of multiple clinical samples with low sample sizes at the same time.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing of RNA, in particular to a low sample size m 6 A high-throughput sequencing method. Background technique [0002] The post-synthetic modification of biological macromolecules plays an important role in many life processes, among which N 6 -Methylated adenosine (m 6 A) is the most abundant post-transcriptional RNA modification in eukaryotic messenger DNA (mRNA). m 6 A modification levels are dynamically reversible in mammalian cells and are regulated by various m 6 Regulation of A-related proteins. Previous studies have shown that m 6 The dynamic adjustment process of A is closely related to the physiological process of life, and m 6 Dysregulation of A has also been shown to lead to some disease-associated pathological changes. m 6 The overall content of A can be obtained by LC-MS after enzymatic digestion of RNA samples, however, due to m 6 A plays an important role in almost al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2525/191C12Q2531/113
Inventor 周翔翁小成秦珊珊韩少卿
Owner WUHAN UNIV