GSM5993351: drug-treated_time_1_Rep_1; Saccharomyces cerevisiae; RNA-Seq
RNA was extracted from 5 A600nm unit/ml of yeast cells, filtered at time 0 and then every hr up to 6 hrs using a membrane filter, as described previously for amino acid extraction (Hepowit et al. 2021). Filtered cells were washed once with 5 ml of ice-cold nanopure water and the filter was quickly transferred to a chilled 1.5 ml microfuge tube containing 0.5 ml cold nanopure water. Tubes were vortexed 10 sec followed by centrifugation for 15 sec. Supernatant fluid (450 μl) was transferred to a new tube and was frozen in a dry-ice EtOH bath followed by storage at -80°C. Cold acid-washed glass beads (300 μl, 0.5 mm dia.) were added to a frozen cell pellet followed by addition of 300 μl of RLT buffer (RNAeasy mini kit (Qiagen, Germantown, Maryland). Tubes were vortexed 5 min at room temperature and placed on ice for 1 min. This cycle was done 4 times followed by addition of 300 μl of ice-cold RLT buffer, mixing, and centrifugation at 13,000xg for 2 min at room temperature. Supernatant fluid (450 μl) was transferred to a new microfuge tube and then mixed with 1 ml of 70% EtOH before transfer to a RNeasy spin column and processed according to the manufacture's instructions. Processed samples were frozen in a dry-ice EtOH bath and stored at -80°C. Libraries were constructed with Illumina's 'TruSeq Stranded mRNAseq Sample Prep kit' at the Roy J. Carver Biotechnology Center at the University of Illinois.