Protocol for using TRIBE to study RNA-protein interactions and nuclear organization in mammalian cells

Abstract

Summary: Targets of RNA-binding proteins discovered by editing (TRIBE) determines RNA-proteins interactions and nuclear organization with minimal false positives. We detail necessary steps for performing mammalian cell RBP-TRIBE to determine the targets of RNA-binding proteins and MS2-TRIBE to determine RNA-RNA interactions within the nucleus. Necessary steps for performing a TRIBE experiment are detailed, starting with plasmid/cell line generation, cellular transfection, and RNA sequencing library preparation and concluding with bioinformatics analysis of RNA editing sites and identification of target RNAs.For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020).

Keywords

• Bioinformatics
• Flow Cytometry/Mass Cytometry
• Gene Expression
• Genomics
• Molecular Biology
• RNAseq