GSM5741642: B_DMSO_S64; Saccharomyces cerevisiae; RNA-Seq
Forty-five ml of S. cerevisiae cell culture (at an OD of 1.0) was harvested, washed in ice-cold RNase-free water twice, and stored at -80°C. In parallel, 5 ml of logarithmically growing S. pombe WT cells was harvested (at an OD of 0.4) for spike-in normalization, washed in ice-cold RNase-free water twice, and stored at -80°C. A cell pellet from S. cerevisiae, and one from S. pombe were combined in the same tube and allowed to thaw on ice. Thereafter, 1 ml of cold Trizol (Thermo Fisher Scientific, 15596026) and cold glass beads (Sigma, G8772) were added to just about cover the liquid. The tubes were vortexed for 8 minutes, and set on ice afterwards. One ml of cold Trizol was again added and a brief vortex was performed. The liquid was extracted to 1.7 ml Costar tubes. The samples were centrifuged at 12000 g for 10 minutes at room temperature, and supernatants were transferred to new Costar tubes. To the samples, 200 μl of chloroform was added (Sigma, 1024451000), and the tubes were shaken by hand for 15 seconds. The samples were allowed to settle for 2 minutes before being centrifuged at 12000 g for 15 minutes at 4°C. The aqueous phase was collected, and chloroform extraction was performed a second time. Thereafter, 500 μl of 2-propanol (Sigma, 1096341000) was added to the samples. The samples were inverted 7 times, and allowed to precipitate for 10 minutes at room temperature. The precipitates were pelleted at 7500 g for 5 minutes at 4°C, and the pellets were washed once with 1 ml of 70% ethanol, airdried, and resuspended in 25 RNase/DNase-free water. Twenty-five mg of extracted RNA was then treated with 10 units of DNase I (Sigma, 4716728001) for 15 minutes at room temperature. Large ribosomal rRNA molecules were then depleted from the samples using the RiboMinus™ Transcriptome Isolation Kit (Thermo Fisher Scientific, K155003) according to the manufacturer's instructions. Samples were then prepared for sequencing using the NEBNext Ultra II Directional RNA Library Prep (NEB, E7760S) according to the manufacturer's protocol.