Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)
By Eric L. Van Nostrand, G. Pratt, Alexander A. Shishkin, C. Gelboin-Burkhart, Mark Y. Fang, B. Sundararaman, S. Blue, Thai Nguyen, Christine Surka, Keri Elkins and 4 more. Nature Methods, 2016
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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. 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 https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, 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.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/6353d19a4bf6b314be0cae15b476363a4d49216f
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In Molecular Biology
Citations in the corpus
- The stress granule transcriptome reveals principles of mRNA accumulation in stress granules (2017, neutral): “All eCLIP data were analyzed from a previous report, which examined eCLIP targets of over 80 proteins (Van Nostrand et al., 2016).”
- RNA-Binding Proteins in Cancer: Old Players and New Actors. (2017, background): “Very recently, advances to the iCLIP methodology concerning the efficiency of key biochemical reactions have been developed, termed enhanced (e)CLIP and infrared (ir)CLIP [227,228].”
- STREME: Accurate and versatile sequence motif discovery (2020, methodology): “As with the DNA (ChIP-seq) experiments above, we compare motif discovery algorithms run on data from an in vivo assay—eCLIP data from ENCODE [17]—using reference motifs for the same RBPs derived using an in vitro assay— RNAcompete [11].”
- The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation (2020, methodology): “eCLIP was performed as described previously with minor modifications (23).”