An improved iCLIP protocol

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.

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

“…Ribosome footprints from three biological replicates of both TDP-43-knockdown control samples were generated and purified as described, using a sucrose cushion 49 and a customized library preparation method based on revised iCLIP 50 . No ribosomal RNA depletion step was performed, and libraries were sequenced on an Illumina Hi-Seq 4000 machine (SR100).…”

“…Plasmid (1.25 μg) was used for each dish, measured via Nanodrop (Thermo Fisher Scientific), combined with 2.5 μl of Lipofectamine 3000 and P3000 reagent diluted in 250 μl (2 × 125 μl) of Opti-MEM I following the manufacturer protocol (Thermo Fisher Scientific). Cells were UV crosslinked on ice and subjected to iCLIP analysis following the iiCLIP protocol 50 . In brief, medium RNase I was added to cell lysate for RNA fragmentation.…”

Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells

“…After 24 hours cells were irradiated 1x with 254 nm UV light (150 mJ/cm2). iCLIP libraries were carried out following an improved iCLIP protocol [34]. Cells were lysed with 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630 (Sigma I8896), 0.1% SDS and 0.5% sodium deoxycholate lysis buffer.…”

“…PTBP1-RNA complexes were precipitated with 2 µg of anti-PTBP1 antibody (ThermoFisher clone 1 cat#:32-4800) and 20 µl of Protein A/G beads (Pierce cat#:88802) overnight rotating at 4°C. RNA isolation and cDNA library preparation was carried out as described in Lee et al [34]. using the L3-IR-App adapter to visualise and retrotranscribe the RNA crosslinked to PTBP1.…”

Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

“…However, despite the obvious power of direct experimental identification of miRNA targets and recent efforts to improve the efficiency of these approaches in capturing additional chimeric molecules (Bjerke and Yi, 2020; Gay et al, 2018), widespread adoption of these methods remains limited, as both the poor recovery of AGO-crosslinked RNA as well as the low fraction of chimeric reads make it difficult to deeply profile the interactome of a miRNA of interest at a reasonable cost. We reasoned that the recent development of improved CLIP methods that increase the recovery of protein-bound RNA by more than a thousand-fold over prior CLIP methods (Lee et al, 2021; Van Nostrand et al, 2016; Zarnegar et al, 2016) represented an opportunity to expand the utility of chimeric CLIP approaches.…”