GSM8781981: Yeast W303 REP2; Saccharomyces cerevisiae; RNA-Seq
RNA was isolated from cell pellets using Direct-zol™ RNA Miniprep kit (Zymo research #R2051) according to manufacturer instructions. In brief, cells were lysed using Trizol® and DNA was digested using provided DNAse I. After three washing steps, the RNA was eluted. RNA concentration was measured with a NanoDrop™ (ThermoFisher) and stored at -80 °C or used immediately for library preparation. Sequencing libraries were created using the Smart-seq2 protocol (Picelli et al. 2014). In brief, 100 ng of RNA were used for reverse transcription and subsequent preamplification PCR. PCR product was purified using Ampure XP beads at 1:0.8 ratio. Concentration of purified cDNA was measured using Qubit (Thermofisher), and quality was checked using Qsep Bio-Fragment Analyzer (NIPPON Genetics).  4 ng of cDNA was tagmented using Tn5 (Nextera) barcoded and PCR amplified. PCR product was purified with Ampure XP beads at a ratio of 1:0,9. Library concentration was measured using Qubit and quality was checked using Qsep Bio-Fragment Analyzer. Libraries were sequenced on an Illumina Nextseq 500 at 75bp single-end reads.