2.10. Cell Culture and Cytoplasmic Lysate Preparation

MCF-7 cells (originally from ECACC, Salisbury, UK) were maintained in DMEM medium. All medium solutions were supplemented with 10% fetal bovine serum, non-essential amino acids (Gibco-BRL, Thermo Fischer Scientific), 100 U/mL of penicillin G, 0.1 mg/mL of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) and the cells were maintained at 37 °C in a 5% carbon dioxide atmosphere. Genotoxic stress was generated by addition of doxorubicin to a final concentration of 0.5 μg/mL. Approximately 4 × 10⁷ MCF7 cells were used per extract for one RNA-affinity chromatography procedure. The cells were washed with PBS buffer and then they were collected by centrifugation at 1000 rpm. The pellet was resuspended in 5 pellet volumes of CE buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% (v/v) NP40, 1 mM DTT, and 1 mM PMSF, pH 7.6) and incubated on ice for 3 min. Then cell lysate was centrifuged at 1000–1500 rpm for 4 min and the cytoplasmic extract was collected.