title: GSM9223052: WY23, 35% fermentation, rep-2; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: RNA was extracted using a hot acid phenol protocol. Frozen cell pellets were resuspended in 400 µL TES solution. An equal volume of acid phenol was added to each tube and vortexed. Tubes were incubated in a 65°C water bath for 1 hour with vortexing every 10-12 minutes. Phases were separated by microcentrifugation in the cold for 5 minutes. The aqueous layer was extracted into a new tube. A second round of acid phenol extraction was conducted followed by a chloroform extraction. RNA was precipitated with 300 µL of 4 M LiCl in dry ice for 20 minutes followed by 5 minutes of high-speed centrifugation at 4°C. The pellet was washed with 500 µL ice cold 70% ethanol and dried for 10 minutes at 37°C. RNA pellets were resuspended in DEPC treated water and stored at -20°C. Contaminating genomic DNA was removed using the DNase Max Kit (Qiagen). RNA sample quality was assessed using an Experion automated electrophoresis system with a HighSens chip (Bio-Rad). cDNA libraries were prepared using the Illumina Stranded mRNA Prep protocol (Illumina). A total of 150-200 ng of total RNA per sample and 13 cycles of amplification were used. The Illumina Dual Index Adapters set A (Integrated DNA Technologies) was ligated to the cDNA. cDNA libraries were quantified using fluorometry with PicoGreen. The integrity of the cDNA libraries was determined using an Agilent BioAnalyzer DNA chip. A KAPA qPCR was used to get precise quantification prior to loading samples for sequencing. The pooled libraries were run with the 2x150 bp protocol on one lane of an SP flow cell in an Illumina NovaSeq.