PAR-CLIP of PTBP1 in HeLa cells
HeLa cells were transfected with a plasmid expressing PTBP1 Tagged at the C-terminus with a dual 6His-biotin tag. Cells were grown for 14 hrs in the presence of 4-thiouridine, media was then removed, and cells were irradiated for one minute with a 365nm UV-LED light source. Cross-linked cells were removed by scraping in PBS, centrifuged; pellets were frozen at -80°C. Pellets from ten 15cm plates of cells were resuspended in 7.5 ml of 300 mM NaCl, 0.5% NP-40, 50 mM NaPO4 pH 7.2, 10 mM immidazole, 6 M Guanidine HCl, Protease inhibitor Cocktail VII and the fusion protein was purified by tandem affinity chromatography and cross-linked RNA used for library construction and sequencing by Illumina HiSeq.