High-throughput sequencing method for efficient research of RNA interaction interactome and application of high-throughput sequencing method

A high-throughput and efficient technology, applied in the field of RNA interactomics, which can solve the problems of underestimation, difficult detection of RNA interactions, and mixing.

Active Publication Date: 2019-09-06
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the RNA-based ortho-crosslinking technology can directly reflect the RNA-RNA interaction and the secondary structure of RNA in vivo, it also has some disadvantages.
The first is that when psoralen is cross-linked, it has a bias towards base selection, and it tends to fix adjacent relative pyrimidine bases, especially uracil, which may cause G-C enriched regions in the results to be underestimated
The second is that the propo

Method used

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  • High-throughput sequencing method for efficient research of RNA interaction interactome and application of high-throughput sequencing method
  • High-throughput sequencing method for efficient research of RNA interaction interactome and application of high-throughput sequencing method
  • High-throughput sequencing method for efficient research of RNA interaction interactome and application of high-throughput sequencing method

Examples

Experimental program
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Effect test

Embodiment 1

[0268] 1. Design reverse complementary probes for U6 snRNA using CIRDES design rules

[0269] First query the human U6snRNA sequence (>NC_000015.10:c67840045-67839940, Homo sapiens chromosome 15, GRCh38.p12 Primary Assembly) through the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ), the result See Table 1; then design according to the above-mentioned CIRDES probe design principles, because the length of the RNA fragment is less than 200nt, design two probes, the results are shown in Table 1.

[0270] Table 1: U6 snRNA sequence and U6 probe sequence designed by CIRDES technology

[0271]

[0272] 2. CIRDES technology enriches U6 snRNA and its interacting RNA

[0273] Culture HepG2 cells in a 15cm dish according to the above method, and then use 2% (v / v) formaldehyde solution, according to 1×10 7 Cells: 10mL formaldehyde solution was cross-linked and fixed, and after 80 cycles of ultrasonic homogenization, the nucleic acid in the cell lysate was fragmented into a lengt...

Embodiment 2

[0292] With reference to the experimental procedure of Example 1, the difference is that the concentration and consumption of crosslinking agent formaldehyde are different, and it is divided into the following five groups of experiments:

[0293] (1) No cross-linking agent formaldehyde (named: -Formaldehyde);

[0294] (2) Use 1% (v / v) formaldehyde solution, according to 1×10 7 Each cell: 2.5mL formaldehyde solution cross-linked and fixed (named: 1%-Formaldehyde-2.5mL);

[0295] (3) Use 2% (v / v) formaldehyde solution, according to 1×10 7 Each cell: 2.5mL formaldehyde solution cross-linked and fixed (named: 2%-Formaldehyde-2.5mL);

[0296] (4) Use 2% (v / v) formaldehyde solution, according to 1×10 7 Cells: 10mL formaldehyde solution cross-linked and fixed (named: 2%-Formaldehyde-10mL);

[0297] (5) Use 3% (v / v) formaldehyde solution, according to 1×10 7 One cell: 2.5mL formaldehyde solution cross-linked and fixed (named: 3%-Formaldehyde-2.5mL).

[0298] The abundance of U4R...

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Abstract

The invention discloses a high-throughput sequencing method for efficient research of RNA interaction interactome and application of the high-throughput sequencing method. The method includes steps: (1) arranging DNA probes at intervals of 100bp according to length of to-be-tested RNA; (2) after crosslinking fixation with 1-3% (v/v) of formaldehyde solution, subjecting cells to ultrasonication toobtain cell lysis solution in nucleic acid length range of 100-500nt; (3) adding the DNA probes for hybridization, gathering target RNA, washing, and purifying to obtain RNA fragments; (4) connecting3' terminals of the RNA fragments with RNA joints, performing product reverse transcription to obtain cDNA, connecting the 3' terminal of the cDNA with a DNA joint to obtain a library of the target RNA and RNAs in interaction with the target RAN; (5) amplifying the library, and carrying out detection analysis. The method can be used for library construction of low-content interaction RNAs.

Description

technical field [0001] The invention relates to the field of RNA interactome, in particular to a high-throughput sequencing method for efficiently studying RNA interactome and its application. Background technique [0002] RNA-RNA interactions are fundamental to the process of gene expression. They perform important functions in gene transcription, translation, post-transcriptional and post-translational modification. RNA-RNA interactions include: small nuclear (sn) RNA–snRNA and snRNA–precursor messenger (pre-m) RNA interactions, which are involved in the assembly, catalytic reactions, and disassembly of the spliceosome, which is the exercise The pre-mRNA is processed into the function of mature mRNA by splicing the intron, thus laying the foundation for the functional translation of the subsequent mRNA. In addition, the interaction between aminoacylated trandfer RNAs (tRNAs) and mRNAs is involved in the peptide chain production process during protein translation. The in...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2525/191C12Q2521/525Y02A50/30
Inventor 尹东李耀庭廖建友
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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