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RNA Detection pp 427–454Cite as

Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein–RNA Interactions

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1649))

Abstract

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP–RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.

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Acknowledgments

The iCLIP protocol described is based on previous versions developed in the Ule and Konig labs by many individuals. I would like to extend thanks to all those who have contributed to the method development over the years. I would also like to thank Prof. Jernej Ule for critical reading of the chapter, and Prof. Jernej Ule and Flora Lee for providing the adapter oligo used in this protocol. This work is supported by an Edmond Lily Safra Fellowship to C.R.S.

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Authors and Affiliations

  1. Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK

    Christopher R. Sibley

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  1. Christopher R. Sibley
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Correspondence to Christopher R. Sibley .

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Editors and Affiliations

  1. Developmental Biology Unit, EMBL Heidelberg, Heidelberg, Germany

    Imre Gaspar

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Sibley, C.R. (2018). Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein–RNA Interactions. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_29

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  • DOI: https://doi.org/10.1007/978-1-4939-7213-5_29

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7212-8

  • Online ISBN: 978-1-4939-7213-5

  • eBook Packages: Springer Protocols

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