GSM9436301: Targeted SHAPE-MaP analysis of MTX targets in MDA-MB-231 cells, 5 mM MTX, 2A3-treated; Homo sapiens; OTHER
Cells were lysed by direct addition of 2 mL ice-cold TRIzol™ Reagent (cat. 15596018, ThermoFisher Scientific). RNA samples in TRIzol™ were mixed with 0.2 volumes chloroform (cat. 319988, Merck) by vigorous vortexing for 15 sec, after which samples were allowed to sit at room temperature for 2 min, before centrifugation at 12,500g for 15 min (4°C). After phase separation, the upper aqueous phase was transferred to a clean 2 mL tube and mixed with 1 volume 2-propanol (cat. 190764, Merck) by vigorous vortexing for 15 sec. Samples were allowed to sit at room temperature for 10 min, before centrifugation at 17,000g for 10 min (4°C). Supernatant was discarded and the RNA pellet was washed twice in 75% ethanol. The RNA pellet was then allowed to air-dry for 10 min, before resuspension in nuclease-free water, pre-warmed at 70°C. Residual gDNA was removed by digestio I at 37°C for 30 min.