GSM8193496: WT_FT671_NEUR_Rep2; Homo sapiens; RNA-Seq
RNA was isolated using the TriPure isolation reagent following the manufacturers protocol for RNA-isolation. The medium was removed from the cells and the TriPure isolation reagent was added directly onto the cells (10cm dish; 5ml TRIPURE reagent,12 wells; 0.5ml TRIPURE reagent). After 5 minutes of incubation the cell lysate(1ml) was transferred into a 1.5-ml Eppedorf tube, after addition of 0.2ml chloroform the tubes were vigorously shaken and incubated at room temperature for 15minutes. Samples were centrifuged for 15 min at 12,000g at 4°C, thereafter the aqueous phase was transferred to a new 1.5-ml Eppedorf tube to which 0.5ml isopropanol was added, mixed and incubated at room temperature for 10minutes, followed by centrifugation 10 min at 12,000g at 4°C. The pellet containing the precipitated RNA was washed twice with 1ml 75% ethanol, dried for 3minutes using the SpeedVac and resuspended in 100μl of nuclease-free water. RNA concentrations and purities were determined using the Nanodrop spectrophotometer. PolyA-total mRNA sequencing by BGI