GSM5155545: HH117 Rep 3 X117_2; Saccharomyces cerevisiae; RNA-Seq
For obtaining RNA, each line was pulled onto solid YPD and allowed to grow for two days at 30C, and then three individual colonies (biological replicates) were used to inoculate three 3ml liquid YPD cultures (no agarose) of each line. Liquid cultures were incubated on a rotator at 30C for 24 hours, before being diluted into 50ml YPD and allowed to grow on a shaker at 30C for 6 hours. Optical density (OD) measurements were taken to ensure all cultures were in the same log growth phase. Cells were then pelleted, and RNA was extracted from each replicate using the MasterPure Yeast RNA Purification Kit (Epicentre). Integrity, concentration, and quality of RNA samples were assessed using a Qubit (Thermo Fisher Scientific). Libraries were prepared using the Illumina Stranded RNAseq Kit and were sequenced at the Georgia Genomics and Bioinformatics Core (https://dna.uga.edu/) on the Illumina NextSeq (75 cycles) single end 75bp reads High Output flow cell. Samples were multiplexed and split across two sequencing lanes.