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Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)

Nature Methods volume 13, pages 508514 (2016) | Download Citation


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, 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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The authors would like to thank members of the Yeo lab (particularly S. Aigner and S. Markmiller) as well as colleagues J. Van Nostrand, Y. Kobayashi, B.R. Graveley and C.B. Burge for critical reading of the manuscript, and M. Blanco with early method development. This work was supported by grants from the US National Institutes of Health (HG004659, U54HG007005 and NS075449 to G.W.Y.), and by the US National Institutes of Health Director's Early Independence Award (DP5OD012190) and funds from the California Institute of Technology to M.G. We would also like to thank Ionis Pharmaceuticals for sharing reagents. E.L.V.N. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2172-13). G.W.Y. is an Alfred P. Sloan Research Fellow. G.A.P. is supported by the National Science Foundation Graduate Research Fellowship.

Author information


  1. Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  2. Stem Cell Program, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  3. Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  4. Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.

    • Gabriel A Pratt
    •  & Gene W Yeo
  5. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.

    • Alexander A Shishkin
    • , Christine Surka
    •  & Mitchell Guttman
  6. Ionis Pharmaceuticals, Carlsbad, California, USA.

    • Frank Rigo
  7. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    • Gene W Yeo
  8. Molecular Engineering Laboratory, A*STAR, Singapore.

    • Gene W Yeo


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E.L.V.N., A.A.S., M.G., and G.W.Y. conceived the study. E.L.V.N., A.A.S., and C.S. developed the eCLIP methodology. E.L.V.N., C.G.-B., and S.M.B. performed 293T eCLIP and RBFOX2 knockdown experiments. F.R. provided antisense oligonucleotides (ASOs) and M.Y.F. performed ASO experiments. C.G.-B., B.S., S.M.B., T.B.N., K.E., and R.S. performed K562 and HepG2 eCLIP experiments. E.L.V.N. and G.A.P. performed computational analyses. E.L.V.N. and G.W.Y. wrote the manuscript.

Competing interests

F.R. is a paid employee of Ionis Pharmaceuticals.

Corresponding author

Correspondence to Gene W Yeo.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Table 3, and Supplementary Protocol 1 and 2

Excel files

  1. 1.

    Supplementary Table 1

    Public CLIP dataset listing and associated read mapping values.

  2. 2.

    Supplementary Table 2

    eCLIP experiments deposited at the ENCODE Data Coordination Center, and associated read mapping values.

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