PT  - JOURNAL ARTICLE
AU  - Flora C. Y. Lee
AU  - Anob M. Chakrabarti
AU  - Heike Hänel
AU  - Elisa Monzón-Casanova
AU  - Martina Hallegger
AU  - Cristina Militti
AU  - Federica Capraro
AU  - Christoph Sadée
AU  - Patrick Toolan-Kerr
AU  - Oscar Wilkins
AU  - Martin Turner
AU  - Julian König
AU  - Christopher R. Sibley
AU  - Jernej Ule
TI  - An improved iCLIP protocol
AID  - 10.1101/2021.08.27.457890
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.08.27.457890
4099  - http://biorxiv.org/content/early/2021/08/27/2021.08.27.457890.short
4100  - http://biorxiv.org/content/early/2021/08/27/2021.08.27.457890.full
AB  - 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 StatementC.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.