GSM5381174: RNA_WT_D2_pe; Saccharomyces cerevisiae; RNA-Seq
Cells were first quenched in TCA (10% final) and washed with acetone. Cell lysis was performed by bead-beating in urea buffer containing 6 M urea, 1% SDS, 50 mM Tris-HCl pH 7.5, 50 mM NaF, 5 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail (Roche). Supernatants of the lysates were collected after heating for 10 min at 75°C. RNA isolation of one OD600 unit of cells was performed using MasterPure yeast RNA purification kit (Epicenter) following the manufacturer's protocol. RNA concentration was determined by A260. 1 mg RNA was reverse transcribed to cDNA using Superscript III Reverse Transcriptase from Invitrogen. Real-time PCR was performed in triplicate with SYBR GreenSuper mix from BioRad.