GSM5332829: YPW3286_rep4 [120010]; Saccharomyces cerevisiae; RNA-Seq
Frozen cell pellets were resuspended in 700uL of a lysis buffer containing beta-mercaptoethanol and transferred to a plate containing ~250uL of acid-washed 425-600um beads. Beaded plates were beaten 10x for 1min each with 1min on ice in between. Plates were spun down at 3000rpm at 4C for 4min, and 400uL of lysis supernatant were removed and transferred into a new 96 well plate containing oligodT magnetic beads (Dynabeads mRNA DIRECT Kit, Ambion, cat# 61011). Beads and lysate were incubated at room temperature with agitation for 5 minutes, and then placed on a magnetic stand. Beads were then washed according to the Dynabeads mRNA direct kit user guide. After extraction, several representative samples were run on Agilent Bioanalyzer to check RNA quality and concentration before moving on to library preparation. In addition, selected wells were tested for the presence of NAT or KAN resistance markers present in distinct strains and controls by RT-PCR and visualized on a gel to ensure well-cross contamination had not taken place at any step previous to library prep.  RNA sequencing libraries were prepared using a modified, ⅓ volume reaction TruSeq RNA Sample Preparation v2 Guide, and using multiplexable adapters from the TruSeq RNA CD Index Plate (cat# 20019792).