Journal: Genome Biology
Article Title: Insights into the design and interpretation of iCLIP experiments
Figure Lengend Snippet: An overview of methods and experiments. a A simplified schematic of the iCLIP protocol [ 17 ]. Before, cells or tissues are irradiated with UV light, which creates covalent bonds between proteins and RNAs that are in direct contact (step 1). After lysis, the crosslinked RNA is fragmented by limited concentration of RNase I and RNA fragments are then co-immunoprecipitated with the RBP (step 2), followed by ligation of a 3′ adapter (step 3). After SDS-PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments (step 5). Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). The peptide that is on the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the crosslink site. Therefore, two types of cDNAs are generated: truncated cDNAs and readthrough cDNAs. In iCLIP, the cDNA library is prepared in such a way that both truncated and readthrough cDNAs are amplified (step 7). After PCR amplification and sequencing (step 8), both truncated and readthrough cDNAs are present. b Table summarising the experiments used in this study. 4SU using 4SU combined with UV-A crosslinking, RNase optimised RNase digest conditions including antiRNase inhibitor and increased RNase I concentration, dephospho omitting 3′ dephosphorylation
Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).
Techniques: Irradiation, Lysis, Concentration Assay, Immunoprecipitation, Ligation, SDS Page, Purification, Generated, cDNA Library Assay, Amplification, Polymerase Chain Reaction, Sequencing, De-Phosphorylation Assay