For cells with a ~2 h dissolved oxygen period, 32 samples (the pellet from 0.5 ml with the supernatant removed) were removed every 4 min, immediately frozen in liquid nitrogen, and store at -80 C.
Growth protocol
CEN.PK113-7D (MATa), grown overnight in 20 ml YPD at 275 rpm and 30C, was inoculated into the fermentor. The fermentor maintained the pH, agitation speed, aeration, and temperature at 4.0, 750 rpm, 150 ml/min, and 30 oC, respectively. Cells were grown in either the B. Braun Biotech Biolab CP (Aylesbury, Buckinghamshire, UK) at 650 ml or the New Brunswick Scientific Bioflo 110 Fermentor/Bioreactor (Edison, NJ) at 850 ml volume. Continuous flow was started after 24-30 h by pumping in fermenter media (19.25 g/l D(+)glucose monohydrate, 5 g/l (NH4)2SO4, 2 g/l KH2PO4, 0.5 g/l MgSO4, 1 g/l yeast extract, 0.2 ml/l antifoam A, 0.1 g/l CaCl2, 0.02 g/l FeSO4, 0.01 g/l ZnSO4, 0.005 g/l CuSO4, 0.001 g/l MnCl2, and 1 ml/l 75% H2SO4) at a dilution rate of 0.086/h. Media with excess cells was pumped out of the fermentor to maintain a constant fermentor volume. The media was maintained at pH 4.0 with 2.5 N NaOH. The dilution rate was adjusted to 0.06-0.086/h to maintain the period.
Extracted molecule
total RNA
Extraction protocol
RNA was purified from the cells and DNA was digested with DNase 1 using the RiboPureTM Yeast kit (Ambion, Austin, TX) according to the manufacturer’s instructions, except the cells were lysed in a minibead beater (BioSpec Products, Bartlesville, OK) rather than by vortexing. The isolated RNA was analyzed for concentration and purity with the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Biotin
Label protocol
GeneChip Expression 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and oligo(dT) primers containing T7 RNA polymerase promoter was used to synthesize double-stranded cDNA from RNA adjusted to 1 mg/ml for each sample. Then, biotinylated cRNA was synthesized using the GeneChip IVT labeling kit from Affymetrix. After framentation to 35-200 bases according to the Affymetrix protocol, 5 micrograms was hybridized to Affymetrix GeneChip Yeast Genome 2.0 Array (Santa Clara, CA) for 16 h at 45°C. The arrays were washed and stained with streptavidin-phycoerythrin using an Affymetrix Fluidics Station 450.
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
GeneChips were scanned using an Affymetrix GeneArray scanner according to the manufacturer’s instructions.
Description
B573_yeast-R10
Data processing
MicroArray Suite 6.1 and GCOS were used to quantify and analyze results. The submitted files show raw and unnormalized data adjusted for hybridization efficiency using biotinylated Escherichia coli standards.
