Discovery of interaction sites between RNA-binding proteins (RBPs) and their RNA targets plays a critical role in enabling our understanding of how these RBPs control RNA processing and regulation. Cross-linking and immunoprecipitation (CLIP) provides a generalizable, transcriptome-wide method by which RBP/RNA complexes are purified and sequenced to identify sites of intermolecular contact. By simplifying technical challenges in prior CLIP methods and incorporating the generation of and quantitative comparison against size-matched input controls, the single-end enhanced CLIP (seCLIP) protocol allows for the profiling of these interactions with high resolution, efficiency and scalability.
Researchers from the University of California San Diego present a step-by-step guide to the seCLIP method, detailing critical steps and offering insights regarding troubleshooting and expected results while carrying out the ~4-d protocol. Furthermore, they describe a comprehensive bioinformatics pipeline that offers users the tools necessary to process two replicate datasets and identify reproducible and significant peaks for an RBP of interest in ~2 d.
Overview of the seCLIP protocol
a, UV cross-linking of cultured cells or tissue (Steps 1–4). b, Cells are lysed, and RNA is partially digested (Steps 8–11). c, The target protein is purified by using antibody-coupled magnetic beads (Step 12). d, RBP-RNA complexes are washed and dephosphorylated, enabling ligation of the 3′ linker (Steps 13–22). e, RBP-RNA complexes are denatured from beads and separated by SDS-PAGE, followed by transfer to nitrocellulose and PVDF membranes (Steps 23–27). IP success is visualized via western blot (Steps 28–31), and RBP-RNA complexes can be visualized via RNA biotinylation and streptavidin-HRP (Box 1). Western blot is used as a guide to isolate regions corresponding to RBP-bound RNA fragments. f, RNA is extracted from the membrane via proteinase digestion, SMInput RNA undergoes dephosphorylation and 3′ linker ligation and RNA is converted to cDNA by reverse transcription (Steps 32–56). g, RNA is degraded, and cDNA is purified (Steps 57–59). h, A UMI-containing linker is ligated to the 3′ end of cDNA molecules, followed by cleanup (Steps 60–65). i, cDNA libraries are quantified by qPCR, PCR-amplified and purified before quantification (Steps 66–82). j, Schematic of the final seCLIP library fragment. The unique molecular identifier is displayed in brown and labeled as UMI on the diagram.