GSM8070866: gcn2Δ NT rep 3; Saccharomyces cerevisiae; RNA-Seq
Cells were flash frozen and RNA was collected using a hot phenol extraction method Total RNA integrity was determined using Agilent Bioanalyzer. Library preparation was performed with 5 to 10 µg of total RNA with a Bioanalyzer RNA Integrity Number (RIN) greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer by heating at 94 degrees for 8 minutes. Fragments were reverse transcribed using SuperScript III RT enzyme (ThermoFisher Scientific) and random hexamers, as per manufacturer's instructions, to yield cDNA. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.