GSM6793335: SF3B1 in vivo iCLIP, HeLa, rep2; Homo sapiens; RIP-Seq
In vivo iCLIP is used to study protein-RNA interactions on individual nucleotide resolution by means of UV crosslinking and immunoprecipitation (PMID: 20601959). For the U2AF2 in vivo iCLIP, data from two iCLIP experiments were combined. The first U2AF2 iCLIP  and PTBP1 iCLIP were performed as previously described in (PMID: 24184352 ). The second U2AF2 iCLIP as well as in vivo iCLIPs for FUBP1, SF1, and SF3B1, were performed using the iCLIP2 protocol as described in (PMID: 31610236). Briefly, HeLa cells were irradiated with 150  mJ/cm2 in a UV-C crosslinker to covalently bind the RNA-binding proteins to the bound nucleic acids. For FUBP1 in vivo iCLIP, cross-linking was achieved by 4sU-mediated cross-linking. HeLa cells were 4sU-labelled by adding a 0.1 M 4-Thiouridine (4sU, Sigma) in DMSO to a final concentration of 100 μM/10 cm cell culture dish. Cells were incubated for 16 h at 37 °C, 5% CO2, under exclusion of light. After incubation, the cells were moved onto ice, shielded from light and irradiated at 365 nm, 800 mJ. During subsequent cell lysis, RNA was partially digested to create 50-200 nt long fragments. Immunoprecipitation of the investigated proteins was performed with antibodies listed in Table S9. The anti-PTBP1 Antibody was a kind gift of Chris Smith (PMID: 17679092). Radioactive labelling at the 3' end of the precipitated RNA enables visualisation of the RNP-complex by SDS-PAGE and transfer to a nitrocellulose membrane. After recovery of protein-RNA complexes from the membrane, a proteinase K digestion results in protein-free RNA. cDNA was synthesised by reverse transcription which stops at the cross-linked site, leading to truncation reads in the sequencing. The cDNA is cleaned twice with MyONE beads. PCR amplification and ProNex size selection are performed to amplify and purify the library, respectively. In vivo iCLIP libraries (except PTBP1 iCLIP libraries) were sequenced on an Illumina NextSeq 500 sequencing machine as 92 nt single-end reads including a 6 nt (or 4 nt in case of the first U2AF2 iCLIP) sample barcode as well as 5+4 nt (or 3+2 nt) UMIs. PTBP1 iCLIP libraries were sequenced on an Illumina GA-II machine (https://doi.org/10.15252/embj.201489852) and then resequenced on an Illumina HiSeq 2000 machine as 50 nt single-end reads including a 4 nt sample barcode as well as 3+2 nt UMIs.