PubReading [118] - Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP) - E. Van Nostrand, G. Yeo et al

<p>As <strong>RNA-binding proteins</strong> (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and <strong>immunoprecipitation</strong> (<strong>CLIP</strong>) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at <a href="https://www.encodeproject.org),">https://www.encodeproject.org),</a> demonstrating that eCLIP enables large-scale and robust <strong>profiling</strong>, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity. - DOI: <a href="https://doi.org/10.1038/nmeth.3810">10.1038/nmeth.3810</a> - 2016</p>