GSM7966627: HEK293T, YB-1 PAR-CLIP, repA; Homo sapiens; OTHER
After crosslinking cells were washed twice with cold PBS, pelleted by centrifugation (500g), lysed with 3 volumes of NP40 buffer (50 mM Hepes-KOH (pH 7.5), 150 mM KCl, 2 mM EDTA, 0.5% NP40, 0.5 mM DTT and a mixture of protease inhibitors (Roche, Switzerland)) and cell debris was pelleted at 13000g for 10 min at 4°C. Further steps of PAR-CLIP were performed according to Danan et al. 2016  (PMID: 26463383). RNA-protein complexes treated with RNase T1, radiolabeled, immunoprecipitated using antibodies against YB-1 (polyclonal rat antibody against 14 a.a. C-terminal peptide of YB-1 (IMTEK, Russia)) and purified using acid-urea electrophoresis. After recovery from Nitrocellulose Membrane and protein digestion, RNA fragments were used for library preparation. PAR-CLIP libraries were obtained  using the ARTseq™ Ribosome Profiling Kit (Epicentre). PAR-CLIP