GSM9346882: H3WT_Rap_biol rep 2; Saccharomyces cerevisiae; RNA-Seq
RNA was isolated using the hot-acid phenol method. Briefly, yeast cells were grown in 10 mL of SC media to OD600=0.8 - 1.0, pelleted, and the supernatant discarded. Pellets were resuspended in 1 mL of ice-cold water, transferred to a clean 1.5 mL microcentrifuge tube, pelleted again, and then the supernatant was removed. Cells were resuspended in 400 μL of TES solution (10 mM Tris HCl (pH 7.5), 10 mM EDTA, and 0.5% SDS), and then 400 μL of acid phenol was added before vortexing vigorously for 10 seconds. Samples were then incubated for 45 minutes at 65 °C with occasional brief vortexing, and then they were placed on ice for five minutes before centrifuging at 4 °C at 15000×rpm for five minutes. The aqueous phase was transferred to a clean 1.5 mL microcentrifuge tube, and the acid phenol treatment was repeated. After isolating the aqueous phase from the second acid phenol extraction, 400 μL of chloroform was added, the samples were vortexed vigorously, and then centrifuged for five minutes at 15,000×rpm at 4 °C. The aqueous phase was transferred to a new tube, ethanol precipitated for 30 minutes and then pelleted at 4 °C. Pellets were washed with ice-cold 70% ethanol, pelleted again, and then resuspended in 40 μL of RNase-free water and incubated at 65 °C for 3-5 minutes to dissolve the RNA pellets. All RNAs were digested with RQ1 DNase (Promega) in a 50 μL total reaction volume, incubated at 37 °C for 45 minutes. RNA was purified by phenol/chloroform extraction, precipitated, and then resuspended in 80 µL of nuclease-free water. QIAseq FastSelect –rRNA Yeast Kit