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.