CRIC-seq protocol for in situ profiling of proximal RNA-RNA contacts associated with RNA-binding proteins

Abstract

Summary: RNA-binding proteins (RBPs) can bind and mediate RNA-RNA contacts. However, identifying specific RBP-organized RNA-RNA contacts remains challenging. Here, we present a capture RIC-seq (CRIC-seq) technique to map specific RBP-associated RNA-RNA contacts globally. We describe steps for formaldehyde cross-linking to fix RNA in situ conformation, pCp-biotin labeling to mark RNA juncture, and in situ proximity ligation to join proximal RNAs. We then detail immunoprecipitation to isolate specific RBP-associated RNA-RNA contacts, biotin-streptavidin selection to enrich chimeric RNAs, and library construction for paired-end sequencing.For complete information on the generation and use of this protocol, please refer to Ye et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Molecular Biology
• Systems Biology