RT Journal Article
SR Electronic
T1 An improved iCLIP protocol
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.08.27.457890
DO 10.1101/2021.08.27.457890
A1 Flora C. Y. Lee
A1 Anob M. Chakrabarti
A1 Heike Hänel
A1 Elisa Monzón-Casanova
A1 Martina Hallegger
A1 Cristina Militti
A1 Federica Capraro
A1 Christoph Sadée
A1 Patrick Toolan-Kerr
A1 Oscar Wilkins
A1 Martin Turner
A1 Julian König
A1 Christopher R. Sibley
A1 Jernej Ule
YR 2021
UL http://biorxiv.org/content/early/2021/08/27/2021.08.27.457890.abstract
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.