Global in situ profiling of RNA-RNA spatial interactions with RIC-seq.
Nat Protoc
; 16(6): 2916-2946, 2021 06.
Article
em En
| MEDLINE
| ID: mdl-34021296
ABSTRACT
Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3' end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Análise de Sequência de RNA
Tipo de estudo:
Evaluation_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
China