10. Analysis of Protein–RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing

  1. Dr. Matthias Harbers3,4 and
  2. Prof. Dr. Günter Kahl5,6,7
  1. Julian König1,
  2. Nicholas J. McGlincy2 and
  3. Jernej Ule1

Published Online: 23 JAN 2012

DOI: 10.1002/9783527644582.ch10

Tag-Based Next Generation Sequencing

Tag-Based Next Generation Sequencing

How to Cite

König, J., McGlincy, N. J. and Ule, J. (2011) Analysis of Protein–RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing, in Tag-Based Next Generation Sequencing (eds M. Harbers and G. Kahl), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527644582.ch10

Editor Information

  1. 3

    4-2-6 Nishihara, Kashiwa-Shi, Chiba 277-0885, Japan

  2. 4

    DNAFORM Inc., Leading Venture Plaza 2, 75-1 Ono-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0046, Japan

  3. 5

    Mohrmühlgasse 3, 63500 Seligenstadt, Germany

  4. 6

    University of Frankfurt am Main Biocenter, Max-von-Lauestraße 9, 60439 Frankfurt am Main, Germany

  5. 7

    Frankfurt Biotechnology Innovation Center (FIZ), GenXPro Ltd, Altenhöferallee 3, 60438 Frankfurt am Main, Germany

Author Information

  1. 1

    MRC Laboratory of Molecular Biology, Division of Structural Studies, Hills Road, Cambridge CB2 0QH, UK

  2. 2

    MRC Laboratory of Molecular Biology, Division of Neurobiology, Hills Road, Cambridge CB2 0QH, UK

Publication History

  1. Published Online: 23 JAN 2012
  2. Published Print: 14 DEC 2011

ISBN Information

Print ISBN: 9783527328192

Online ISBN: 9783527644582

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Keywords:

  • protein–RNA interactions;
  • single-nucleotide resolution;
  • iCLIP;
  • next-generation sequencing;
  • antibody preparation;
  • quality controls

Summary

Post-transcriptional regulation of gene expression is controlled by the unique composition and spatial arrangement of RNA-binding proteins (RBPs) on individual transcripts. Therefore, understanding post-transcriptional regulation requires precise and comprehensive binding site maps for RBPs. UV cross-linking and immunoprecipitation (CLIP) is a state-of-the-art technique for generating such maps on a genome-wide scale. However, data complexity is often limited and the resolution of the resulting maps is confined to approximately 30 nucleotides. This, in turn, complicates the identification of individual binding sites. We recently described individual-nucleotide resolution CLIP (iCLIP) – an approach that both increases data complexity and allows binding site detection at single-nucleotide resolution. Here, we present the latest version of our iCLIP protocol, discussing critical aspects and recent modifications.