![]() Tenenbaum, S.A., Carson, C.C., Lager, P.J. Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs. BackCLIP: a tool to identify common background presence in PAR-CLIP datasets. Reyes-Herrera, P.H., Speck-Hernandez, C.A., Sierra, C.A. A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. iCLIP: protein-RNA interactions at nucleotide resolution. Simultaneous generation of many RNA-seq libraries in a single reaction. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. CLIP identifies Nova-regulated RNA networks in the brain. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Nussbacher, J.K., Batra, R., Lagier-Tourenne, C. RNA-binding proteins in Mendelian disease. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at ), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. 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. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function.
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