GSM5696973: Kse3; Saccharomyces cerevisiae; RNA-Seq
Exponentially grown yeast cultures (OD600 = 0,8). were isolated from a 5 ml culture sample (3,000 rpm; 3 min, 4°C), washed with ice-cold AE buffer (50mM sodium acetate pH 5.2, 10mM EDTA pH 8.0), and stored at -80°C. Next, total RNA was extracted using phenol and chloroform. Residual genomic DNA was removed (1h, 37°C) with DNase I (New England Biolabs, Cat # M0303L). Total RNA was further purified with the RNeasy Kit (Qiagen). RNA concentrations were measured with a Qubit Fluorometer (Invitrogen), and RNA quality and integrity examined with a 2100 Bioanalyzer using  RNA 6000 Pico Kit (Agilent Technologies) CDNA libraries were prepared according to the Illumina Script seq manual. From two μg of purified yeast RNA (see above) all rRNA was removed with the Ribo-Zero Gold rRNA Removal Kit Yeast (Illumina, Cat # MRZY1324). Its depletion was confirmed with a 2100 Bioanalyzer RNA 6000 Pico Chip (Agilent Technologies, Cat # 5067-1513). Next, 10ng of the rRNA-depleted RNA was reverse transcribed into cDNA with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina, Cat # SSV21124). cDNA library concentrations were quantified with a Qubit Fluorometer (Invitrogen), and its quality examined on a 2100 Bioanalyzer using the High Sensitivity DNA assay (Agilent Technologies, Cat # 5067-4626). The cDNA library was then deep sequenced in multiplex fashion (4 samples per run) for 76 bases in the paired-end mode on a HiSeq Sequencing System (Illumina) at CIBIO's Next Generation Sequencing Core facility.