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

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...

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 proportion of RNA chimeras obtained by ligase is relatively small, and the reaction efficiency of ligase still needs to be improved

Therefore, low-abundance RNA interactions are difficult to detect

The third is that the proximity junction not only connects adjacent RNA interaction strands, but also connects other random single-stranded RNAs or RNAs themselves, and will be mixed into subsequent sequencing, which may lead to false positives

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