Proximity‐CLIP and Expedited Non‐Radioactive Library Preparation of Small RNA Footprints for Next‐Generation Sequencing

During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity‐CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside‐enhanced protein‐RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP‐bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity‐CLIP along with a simplified non‐radioactive, small‐RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1 : Cell culture, 4SU labeling, proximity biotinylation, and crosslinking Basic Protocol 2 : Cell extraction, streptavidin affinity purification, and on‐beads trypsinization Basic Protocol 3 : RNA footprints cDNA library preparation Support Protocol : Preparation of RNA‐seq libraries from intact RNA