GSM8825763: DMSO2; Homo sapiens; RNA-Seq
Cells were collected in TRIzole Reagent (Life Technologies, MAN0001271) and 200µL of chloroform was added per 1ml of TRIzole Reagent used. Samples were vortexed thoroughly and incubated on ice for 5 minutes before centrifugation at 13,200 RPM for 10 minutes at 4°C. The aqueous phase containing the RNA was collected and the RNA precipitated with 100% isopropanol and incubated on ice for 5 minutes. The RNA pellets were centrifuged at 13,200 RPM for 10 minutes at 4°C. The supernatant was removed and the RNA pellet washed in 70% ethanol, followed by incubation on ice for 5 minutes. After centrifugating the RNA at 13,200 RPM for 5 minutes at 4°C the supernatant was removed, and the RNA pellet briefly air dried. Finally, the RNA was resuspended in RNAse-free H2O and incubated on ice for 20 minutes. RNA concentrations were determined using Nanodrop 3000 and 200ng of total RNA sent for sequencing. Total RNA-Seq libraries were generated from 200 ng of total RNA using Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit and IDT for Illumina RNA UD Indexes, Ligation (Illumina, San Diego, USA), according to manufacturer's instructions. DNA library were amplified using 15cycles of PCR. Surplus PCR primers were further removed by two successive purifications using SPRIselect beads (Beckman-Coulter, Villepinte, France). The final libraries were checked for quality and quantified using Bioanalyzer 2100 system (Agilent technologies, Les Ulis, France).