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

Van Nostrand, Eric L. and Pratt, Gabriel A. and Shishkin, Alexander A. and Gelboin-Burkhart, Chelsea and Fang, Mark Y. and Sundararaman, Balaji and Blue, Steven M. and Nguyen, Thai B. and Surka, Christine and Elkins, Keri and Stanton, Rebecca and Rigo, Frank and Guttman, Mitchell and Yeo, Gene W. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nature Methods, 13 (6). pp. 508-514. ISSN 1548-7091. https://resolver.caltech.edu/CaltechAUTHORS:20160210-225928240

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[img] Image (JPEG) (Supplementary Figure 1: Large-scale iCLIP experiments indicate poor efficiency) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 2: Optional sample pooling strategy and eCLIP computational analysis workflow) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: eCLIP of RBFOX2 improves library efficiency over iCLIP) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: eCLIP improves library efficiency over iCLIP for IGF2BP1 and IGF2BP2) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Reverse transcriptase termination at crosslink sites leaves stereotypical motif frequencies flanking eCLIP sequence reads) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 6: Functional validation of eCLIP binding sites by antisense oligonucleotide (ASO) blocking) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 7: Validation of standard eCLIP conditions across fragmentation conditions) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 8: Paired Size-Matched Input (SMInput) reveals enrichment over common background in CLIP of histone-binding SLBP) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 9: Paired Size-Matched Input (SMInput) reveals enrichment over common background in CLIP of splicing regulator RBFOX2) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 10: Splicing-sensitive microarray analysis identifies RBFOX2-dependent cassette exons) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 11: SMInput-normalization distinguishes significantly enriched eCLIP peaks which contain known binding motifs from clusters depleted in eCLIP which lack motifs) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 12: eCLIP shows high reproducibility across biological replicates) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 13: Scalable RBP target identification with eCLIP) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 14: eCLIP enables distinction between significant binding and common background) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 15: RNA-centric view of RNA binding protein association) - Supplemental Material
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[img] PDF (Supplementary Figures 1–15, Supplementary Table 3, and Supplementary Protocol 1 and 2) - Supplemental Material
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[img] MS Excel (Supplementary Table 1: Public CLIP dataset listing and associated read mapping values) - Supplemental Material
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[img] MS Excel (Supplementary Table 2: eCLIP experiments deposited at the ENCODE Data Coordination Center, and associated read mapping values) - Supplemental Material
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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1038/nmeth.3810DOIArticle
http://www.nature.com/nmeth/journal/v13/n6/full/nmeth.3810.htmlPublisherArticle
http://rdcu.be/g5wwPublisherFree ReadCube access
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887338/PublisherArticle
https://github.com/gpratt/gatk/releases/tag/2.3.2OrganizationCode
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77634OrganizationGene Expression Omnibus
https://www.encodeproject.org/OrganizationK562 and HepG2 eCLIP data sets
ORCID:
AuthorORCID
Guttman, Mitchell0000-0003-4748-9352
Additional Information:© 2016 Macmillan Publishers Limited. Received 10 November 2015. Accepted 16 February 2016. Published online 28 March 2016. 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 Contributions: 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. Code availability. Custom code used is available at https://github.com/gpratt/gatk/releases/tag/2.3.2, and described in Supplementary Protocol 2. Accession codes. All 293T data sets (including SLBP and RBFOX2 eCLIP, RBFOX2 iCLIP, and microarrays profiling RBFOX2 knockdown) have been deposited at the Gene Expression Omnibus (GSE77634). K562 and HepG2 eCLIP data sets have been deposited for public release at the ENCODE Data Coordination Center (https://www.encodeproject.org), with accession identifiers listed in Supplementary Table 2. Competing financial interests: F.R. is a paid employee of Ionis Pharmaceuticals.
Funders:
Funding AgencyGrant Number
NIHHG004659
NIHU54HG007005
NIHNS075449
NIHDP5OD012190
CaltechUNSPECIFIED
Damon Runyon Cancer Research FoundationDRG-2172-13
Alfred P. Sloan FoundationUNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Issue or Number:6
Record Number:CaltechAUTHORS:20160210-225928240
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160210-225928240
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:64400
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:31 Mar 2016 22:18
Last Modified:03 Oct 2019 09:37

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