UV crosslinking of cells and extract preparation

Three 150 × 20 mm tissue culture dishes (Corning, #430,599) of GFP-T-p27(3ʹUTR) HEK293 cells (total 6 × 10⁷ cells) were treated with 1 µg/ml of tet for 48 h before harvesting. To induce the cisplatin response, 20 µM of CP was added to tet-treated cells after 33 h for 15 h. Thereafter, cells were washed twice with 10 ml pre-warmed PBS and after removal of the final rinse, 6 ml of PBS was added. Cells were exposed to UV light for crosslinking of protein-RNA complexes in vivo and cell-free extracts were prepared as described previously [¹⁰]. In brief, cells were exposed on ice to UV light (254 nm) at 150 mJ/cm² in a Stratalinker 1800 (Stratagene) and centrifuged at 250 g for 5 min at 4ºC. The cell pellet was resuspended in 2 ml of lysis buffer (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% Triton X-100, 5 mM DTT, 20 U/ml DNase I (Promega, #M6101), 100 U/ml RNasin (Promega, #N2611), complete EDTA-free protease-inhibitor cocktail (Roche, #11,836,170,001)). Cell lysates were then combined (total ~6 ml) and subjected to three rounds of sonication (Soniprep150, MSE) and cleared by centrifugation at 15,000 g for 10 min at 4°C. Protein concentrations of extracts were determined using the Bradford assay with BSA as a reference standard.