GSM8287758: Sfp1-K655/657R, 2% glucose, rep1; Saccharomyces cerevisiae; RNA-Seq
Total RNA was extracted following the hot-phenol protocol. In brief, cell pellets were resuspended in 400 μL TES solution (10 mM Tris HCl, 10 mM EDTA, and 0.5% SDS). 400 μL acid phenol was added and then vortexed. The cell suspensions were incubated at 65 ˚ C for 30 minutes with occasional, brief vortexing. Top speed centrifuge was performed to separate the aqueous phase. The aqueous phase was transferred and 400 μL chloroform was added followed by another round of vortex and centrifuge. RNAs were precipitated with sodium acetate and 100% ethanol. Initial precipitated RNAs were washed with ice-cold 70% ethanol. RNA pellets were resuspended in 50 μL RNase-free H2O. Reverse transcription was performed following the 2X SYBR Green PCR Master Mix kits protocol. NEBNext Ultra II Directional RNA library and NEBNext Poly(A) - dual index