GSM4984980: SC_AS_WT_drug_2017; Saccharomyces cerevisiae; RNA-Seq
Cultures were split into two 5ml aliquots prior to spinning down at 3850RPM (331rcf) on an Eppendorf 5810R benchtop centerfuge for 3min.  After spinning down, supernatants were poured out and remaining supernatant was aspirated off with a P1000 pipette.   Cells were then flash frozen in liquid nitrogen and stored at  -80°C.  RNA extraction was performed using the hot acid-phenol extraction protocol of (Solís et al. 2016) with the following changes.  Initial cell volume was 5ml instead of 1.5ml and thus the initial spin for collecting the cells was done for 3min at 3850RPM (331rcf) instead of 30s at 13000RPM (15871rcf).  Acid-phenol:chloroform:isoamyl alcohol (IAA) (125:24:1), pH 4.5 (AM9722) was used instead of pure Acid-Phenol because the chloroform aids in the separation of nucleic acid from proteins and lipids, and the IAA prevents foaming.  The spin following heat incubation was performed at room temperature instead of at 4°C.  A second 400µl chloroform wash was included prior to removing the aqueous phase from the phase lock tubes.  22µl of 3M NaOAc, pH5.2 was added instead of 30µl to precipitate the DNA.  Ethanol Precipitation was done overnight instead of for 30 min. 3' Sequencing Libraries were prepared using the Lexogen QuantSeq 3'mRNA-Seq Library Prep kit FWD using dual indices for each sample.  Select libraries were checked for quality on the Bioanalyzer using a High Sensitivity DNA chip.  Library concentrations were calculated using a Qbit 2.0 Fluorometer (Invitrogen), and 2.65 ng per sample were pooled and sequenced on an Illumina Hiseq 4000 sequencer to an average depth of between 245,000 and 5.6 million reads per sample (median 2.53 million).