Identification of RBP binding sites using RNA deaminases.

Abstract

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing. Identification of their binding sites has important implications for their physiological and disease-related functions. Crosslinking and immunoprecipitation, followed by sequencing (CLIP-seq) and its derivatives, are the most commonly used methods to identify RBP binding sites, but are laborious and require a large amount of starting material. Recent advancements harnessing RNA deaminases in fusion to any RBP of interest, allow for the profiling of RBP binding sites from low-input samples in simpler procedures. Among these efforts, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. This chapter describes the detailed protocol for the STAMP method, including plasmid construction, delivery and sorting, library preparation and bioinformatic data analysis.