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An improved iCLIP protocol

View ORCID ProfileFlora C. Y. Lee, Anob M. Chakrabarti, Heike Hänel, Elisa Monzón-Casanova, Martina Hallegger, Cristina Militti, Federica Capraro, Christoph Sadée, Patrick Toolan-Kerr, Oscar Wilkins, View ORCID ProfileMartin Turner, Julian König, Christopher R. Sibley, View ORCID ProfileJernej Ule
doi: https://doi.org/10.1101/2021.08.27.457890
Flora C. Y. Lee
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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  • ORCID record for Flora C. Y. Lee
Anob M. Chakrabarti
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
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Heike Hänel
3Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
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Elisa Monzón-Casanova
4Immunology Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT
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Martina Hallegger
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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Cristina Militti
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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Federica Capraro
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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Christoph Sadée
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
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Patrick Toolan-Kerr
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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Oscar Wilkins
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
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Martin Turner
4Immunology Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT
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Julian König
3Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
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Christopher R. Sibley
5Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, Edinburgh University, 1 George Square, Edinburgh, EH8 9JZ, UK
6Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK
7Department of Medicine, Division of Brain Sciences, Imperial College London, Burlington Danes, London, UK
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Jernej Ule
1The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
2UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, WC1N 3BG, UK
8National Institute of Chemistry, Hajdrihova ulica 19, 1000 Ljubljana, Slovenia
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  • For correspondence: j.ule@ucl.ac.uk
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Abstract

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

Competing Interest Statement

C.R.S is inventor on a patent application covering specific elements of this method (i.e. adapter removal and size-matched input workflow). The other authors declare no competing interests.

Footnotes

  • https://imaps.goodwright.com/collections/1203/

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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An improved iCLIP protocol
Flora C. Y. Lee, Anob M. Chakrabarti, Heike Hänel, Elisa Monzón-Casanova, Martina Hallegger, Cristina Militti, Federica Capraro, Christoph Sadée, Patrick Toolan-Kerr, Oscar Wilkins, Martin Turner, Julian König, Christopher R. Sibley, Jernej Ule
bioRxiv 2021.08.27.457890; doi: https://doi.org/10.1101/2021.08.27.457890
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An improved iCLIP protocol
Flora C. Y. Lee, Anob M. Chakrabarti, Heike Hänel, Elisa Monzón-Casanova, Martina Hallegger, Cristina Militti, Federica Capraro, Christoph Sadée, Patrick Toolan-Kerr, Oscar Wilkins, Martin Turner, Julian König, Christopher R. Sibley, Jernej Ule
bioRxiv 2021.08.27.457890; doi: https://doi.org/10.1101/2021.08.27.457890

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