GSM9859962: KOD7-R2; Mus musculus; RNA-Seq
RNA was extracted using RNAzol RT (Molecular Research Center, Catalog #R4533), a reagent designed for the isolation of total and small RNA from human, animal, plant, bacterial, and viral samples. Cells were washed with calcium- and magnesium-free DPBS and dissociated with TrypLE for 1 min. TrypLE was aspirated, then 1 mL of mESC medium was added, and cells were gently pipetted to detach them from the culture dish. The cell suspension was transferred to a 15-mL conical tube and pelleted by centrifugation at 500 × g for 3 min. The supernatant was removed, and 500 µL of RNAzol RT was added directly to the pellet. The pellet was fully resuspended by pipetting to ensure complete lysis and homogenization, and the lysate was transferred to a 1.5- mL microcentrifuge tube. A total of 0.2 mL of RNase-free water was added to each tube. Samples were capped, shaken vigorously for 15 s, and incubated for 15 min at room temperature. The mixture was centrifuged at 12,000 × g for 15 min at 4 °C, producing a pellet containing DNA, proteins, and polysaccharides, while RNA remained in the upper phase. The supernatant was carefully transferred to a new tube without disturbing the pellet. An equal volume of 100% isopropanol was added to the supernatant to precipitate RNA. Samples were incubated for 10 min at room temperature and then centrifuged at 12,000 × g for 10 min, forming a white RNA pellet. RNA pellets were washed twice with 75% ethanol (0.4–0.6 mL per mL of initial supernatant). Each wash was followed by centrifugation at 8,000 × g for 3 min at room temperature, after which the ethanol was removed by pipetting. The final RNA pellet was resuspended in 20 µL of RNase-free water without air-drying. Samples were vortexed at room temperature for 2 min to ensure complete solubilization. 45 NEB Ultra II RNA, PolyA selected