GSM7706471: 4SU, UVA, 0h, rep2; Homo sapiens; OTHER
proteins were crosslinked to RNA through overnight incorporation of 5µM 4sU and UVA irradiation (150 J/cm2); after irradiation cells were harvested in ice-cold PBS and lysed in iCLIP lysis buffer; samples were homogenized by sonication for 10 cycles (30 s ON, 30 s OFF) with Bioruptor® Plus and protein concentration was quantified by BCA assay; lysates were incubated with Turbo DNAse and RNAse I dilution (1/50) for 3 min at 37°C; protein clean-up for 50 µg of protein was done with SP3 beads in a 1:10 ratio for 1 h at 4°C. iCLIP2 protocol; RNA was dephosphorylated at the 3´end before L3 linker ligation and 5´end radioactive labelling. After western blotting RNA-protein complexes were isolated and digested with proteinase K; reverse transcription of RNA to cDNA; RNA-Hydrolysis before clean-up of cDNA with MyONE Silane beads and adapter ligation; cDNA was amplified and size selected using ProNex® Size-Selective purification system. The library was sequenced on an Illumina NextSeq 500 sequencer with a read length of 150 nt (single-end), an index length of 6nt as well as 5+4-nt unique molecular identifiers (UMIs).