GSM9386701: 425 cells, control, RNaseH1 overexpression, irradiated; Homo sapiens; RNA-Seq
Total RNA was isolated using the TRIzol reagent (Invitrogen). Approximately 1 million cells were lysed by repetitive pipetting in 1 mL of TRIzol. Phase separation was performed by adding 0.2 mL of chloroform (Sigma-Aldrich), followed by centrifugation at 20,000 g for 15 min at 4°C. The top aqueous layer (~500 µL) was transferred to a new tube and the RNA was precipitated overnight at -80°C with an equal volume (500 µL) of 100% isopropanol (Fisher Scientific), 1/10 volume (50 µL) of 3 M sodium acetate (Teknova), and 4 µL of 5 mg/mL glycogen (Thermo). Samples were then centrifuged at 15,000 g for 20 min at 4°C, washed twice with 1 mL of 75% ethanol, and dissolved in 50 µL of nuclease-free water. RNA libraries for RNA-seq were prepared using the KAPA RNA Hyperprep Kit with RiboErase following the manufacturer's instructions