Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)
Open accessAbstract
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.
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Histone clusters served as a quality control for eCLIP assay and have been previously reported target RNAs for METTL3.
“We identified histone clusters, which served as a quality control for eCLIP assay, which have been previously reported target RNAs for METTL3 (Figure S4A) (Van Nostrand et al., 2016).”
From METTL3 uncouples chromatin accessibility from transcription during retinal development (2025) · cites this paper as Van Nostrand et al., 2016
In Results, ‘Input normalization improves CLIP signal-to-noise’ (page not provided), the paper uses SLBP binding to histone RNAs to distinguish true eCLIP signal from false-positive signal, supporting their use as an assay check. The cited paper does not identify histone RNAs as previously reported METTL3 targets; that part of the claim is unsupported by our copy. (LLM, conf=0.87)
Experimental — accuracy not yet measuredMETTL3-eCLIP was performed as described previously (Van Nostrand et al., 2016).
“METTL3-eCLIP was performed as described previously (Van Nostrand et al., 2016).”
From METTL3 uncouples chromatin accessibility from transcription during retinal development (2025) · cites this paper as Van Nostrand et al., 2016
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- Authors
- Van Nostrand, E. L., Pratt, G. A., Shishkin, A. A., Gelboin-Burkhart, C., Fang, M. Y., Sundararaman, B., Blue, S. M., Nguyen, T. B., Surka, C., Elkins, K., Stanton, R., Rigo, F., Guttman, M., & Yeo, G. W.
- Affiliations
- University of California San Diego (US)
- California Institute of Technology (US)
- Ionis Pharmaceuticals (United States) (US)
- Agency for Science, Technology and Research (SG)
- Corresponding author
- Yeo, Gene W
- ORCID
- Venue
- Nature Methods
- Published
- 2016-03-28
- Volume
- 13
- Issue
- 6
- Pages
- 508-514
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4887338
- OpenAlex
- W2307041907
- Cited by
- 1,397
- References
- 6
- Citations by year
- 2026: 124 · 2025: 141 · 2024: 200 · 2023: 125 · 2022: 156 · 2021: 149 · 2020: 155 · 2019: 125 · 2018: 111 · 2017: 77 · 2016: 32 · 2015: 1
- FWCI
- 59.79
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- CaltechAUTHORS (California Institute of Technology) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health (DP5OD012190)
- National Human Genome Research Institute (R01HG004659, U54HG007005)
- National Cancer Institute (P30CA023100)
- National Institute of General Medical Sciences (T32GM007198)
- National Institute of Neurological Disorders and Stroke (R01NS075449)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- RNA-binding proteins, RBP binding sites, ribonucleoprotein complexes, single-nucleotide resolution, HepG2 cells, K562 cells, cancer cell lines, gene expression profiling
- MeSH terms
- Binding Sites, Cross-Linking Reagents, Humans, Ultraviolet Rays, RNA-Binding Proteins, K562 Cells, Gene Expression Profiling, Immunoprecipitation, Photochemical Processes, Hep G2 Cells, Transcriptome
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