S. cerevisiae CEN.PK113-1A (MATa, URA3, HIS3, LEU2, TRP1, MAL2-8c, SUC2) grown in Verduyn mimimal medium with 10 g/L glucose as carbon source, and supplemented with 10 mg/L ergosterol and 420 mg/L Tween 80, in Biostat CT bioreactor. Sample from steady state with 0.5% oxygen provision.
Treatment protocol
none
Growth protocol
Cells were grown in 0.8–1 L of medium in B. Braun Biotech International (Sartorius AG, Germany) Biostat CT (2.5 Lworking volume) bioreactors. Cultures were inoculated to aninitial OD600 nm of c. 0.5, and maintained as batch culturesfor 6–9 h, after which continuous medium feed was startedwhile the cells were still growing exponentially. Chemostatcultures were maintained at D= 0.10 /h, pH 5.0, and30C, with 1.5 volume gas [volume culture]/min (vvm).For cultures that received 20.9% O2 in the gas stream, O2was replaced with the equivalent volume of N2, so that totalgas flow was kept constant for all experiments. Steady-state samples were taken after the cultures had beenin constant conditions for a minimum of four residence times (six generations). Steady states were assessed over fourto nine residence times (six to 13 generations) for constantbiomass production, carbon dioxide evolution and oxygenuptake rates (CER and OUR), alkali utilization, and extracellularmetabolites, as well as constant intracellular metabolites and gene transcription.Cultures that were fed 2.8% or 20.9% O2 were subject tooscillations. To prevent these, c. 5% of the total cellconcentration in the bioreactor was added to the culture ascells in mid-exponential to late exponential phase at thetime when continuous medium feed was started (Zamamiri et al., 2001).
Extracted molecule
total RNA
Extraction protocol
15 ml of cell culture was mixed with 35 ml cold (+4C) 10mM Na-phosphate buffer on ice. After centrifugation for 5 min, 3500 rpm, +4C, cells were frozen in liquid nitrogen and stored at -80C. Total RNA was extracted using QIAGEN RNeasy Mini Kit (QIAGEN, Inc., Valencia, CA) according to the manufacturers instructions with the following modification to the cell lysis step. 5-20 mg dry mass of cells were suspended in 400µl of cold (+4°C) disruption buffer (20mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM MgCl2, 2 mM DTT). 400µl of phenol-chlorophorm (50:50), 5µl 20% SDS and 400 µl glass beads (0.5 mm diameter; Biospec Products) was added. The cells were disrupted with Fastprep machine (Q-Biogene), 2 x 20s, at speed 6. After centrifugation (14000 rpm, 15 min, +4°C), supernatant was used for total RNA extraction. RNA quality was checked with Agilent bioanalyzer (Agilent Technologies).
Label
biotin
Label protocol
Approximately 2 µg of total RNA was processed to produce biotinylated cRNA targets using Affymetrix One-Cycle Target prep protocol.
Hybridization protocol
Hybridisation was done at +45°C overnight (16 h) according to GeneChip Expression Analysis Technical Manual (Affymetrix). GeneChip Fluidics Station 450 was used to wash and stain the arrays.
Scan protocol
GeneChip Scanner 3000 with AutoLoader was used to scan the arrays.
Description
Steady state sample of glucose-limited chemostat with 0.5 % oxygen provision in a series of chemostats with oxygen provision 0,0.5,1,2.8 and 20.9 % oxygen provision. Replicate 3/4.
Data processing
RMA normalised without outlier sample VTT_Ys23_13_1% and excluding S.pombe data and control probes
