Isopropanol

RNA was extracted from differentiated cells, as explained before [12 (link)]. Briefly, having lysed cells with 1 mL Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged with 200 µL chloroform (Chem-supply, Gillman, SA, Australia), total RNA was trapped in an aqueous phase and precipitated by adding 80 μg glycogen (Life Technologies, Mulgrave, VIC, Australia) and 500 μL isopropanol (Chem-supply, Gillman, SA, Australia), followed by an overnight incubation at −20 °C. RNA was finally dissolved in nuclease-free water, and its integrity and concentration were checked by the Agilent small RNA kit and the 2100 Bioanalyzer according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). All samples had RNA integrity number (RIN) values above 8.5.

Cells were lysed directly in the culture plates by the addition of 1 ml Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA). Lysate was transferred to microcentrifuge tubes and 200 μL of chloroform (Chem-supply, Gillman, SA, Australia) was added before centrifugation at 13,000 g at 4 °C for 10 min in accordance with the manufacturer’s instructions (ThermoFisher). The resultant aqueous phase, containing total RNA, was separated from the organic phase and mixed with 80 μg glycogen (Life Technologies, Mulgrave, VIC, Australia) and 500 μL isopropanol (Chem-supply, Gillman, SA, Australia), and incubated at −20 °C overnight for RNA to precipitate. After centrifuge at 9000 g at 4 °C for 30 min, the supernatant was discarded, and the pellet was washed with 75% cold ethanol twice. Finally, the RNA pellet was re-suspended in nuclease-free water and stored at −80 °C. The concentration and purity of extracted RNA was assessed by the Agilent small RNA kit and the 2100 Bioanalyzer according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). The provided system software was used for automatic calculation of RNA integrity number (RIN). All samples had RIN values above 8.5.