title: GSM7031904: RNA_ WT_Replicate_3; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: For RNA-seq library preparation, total RNA was extracted and purified from aliquots of the same snapped-frozen cells described above using hot phenol-chloroform extraction. Five µg of total RNA was randomly fragmented by mixing with 2× alkaline fragmentation solution (2 mM EDTA, 10 mM Na2CO3, 90 mM NaHCO3, pH ≈ 9.3). After incubation for 20 min at 95ºC, fragmentation reactions were mixed with ice-cold stop solution (300 mM NaOAc pH 5.5, 30 µg GlycoBlue (Invitrogen; AM9516)). RNA was precipitated by adding one part of isopropanol followed by the standard protocol of precipitation. Fragment size selection, library generation, and sequencing were carried out using the same protocol described above for RPF library preparation, except the Ribo-Zero Gold rRNA Removal Kit (Illumina; MRZ11124C), was employed to remove rRNA after linker-ligation.