Illumina HiSeq 2500 sequencing; RNA seq of Saccharomyces cerevisiae expressing pathways designed to increase ATP or GTP consumption
RNA seq of Saccharomyces cerevisiae expressing pathways designed to increase ATP or GTP consumption
Cells from culture samples were harvested rapidly by centrifugation (1 min, 4000g) and the cell pellets flash frozen in liquid nitrogen and stored at −80 °C until required. For the glucose grown chemostats, precultures of each strain were prepared by inoculating YNB minimal medium (0.67% w/v Yeast Nitrogen Base (SIGMA) and 0.5% w/v ammonium sulphate) containing 2% glucose with a single colony picked from an agar plate and incubating at 30°C 200 rpm for 24 h. Fermentors (2 L) containing F1 medium (1000 ml) with 0.25% glucose were inoculated with aliquots of the precultures to produce a starting OD600nm absorbance of 0.05, and grown in batch for 24 h (30 °C, 750 rpm stirrer speed, aeration with 1 L min−1 air). Cultures were then switched to continuous mode, maintaining the pH at 4.5 and the dilution rate at 0.11-0.12 h-1. Initial steady-state culture conditions (designated SS1) were deemed to have been established after more than five vessel volume changes and with cultures exhibiting a constant biomass, at which point culture samples were taken for analysis. For induction, DOX was added to the steady-state cultures to produce a final concentration of 5 g/ml. Culture samples were harvested for analysis at 3 (designated T1) , 6 (designated T2) and 9 h (designated T3) after induction, and a final sample taken after the cultures had reached a new steady state (SS2), as determined by the passage of six vessel volume changes and with cultures exhibiting a constant biomass. A similar protocol was followed for the acetate grown chemostat experiments except the initial preculture was performed for 42 h in YNB minimal medium containing 0.5% sodium acetate and 1% glucose. This was used to inoculate batch growth in fermentors containing YNB minimal medium with 0.5% sodium acetate and 0.05% glucose, then switched to continuous mode using only 0.5% sodium acetate as the carbon source and controlling the pH at 5.0 and the dilution rate at 0.07-0.08 h-1. After taking the induced steady state samples, designated SS2, an additional experiment was performed to analyse the response to adding glucose to the respiring, induced cultures. Glucose (40%)  was added to each fermentor (and to the feed for each fermentor)  to produce a final concentration of 1.6%, and culture samples taken at 15 min (designated G1), 30 min (designated G2) and 60 min (designated G3) after glucose addition. For RNA purification, frozen cells were resuspended in trizol™(Invitrogen) and lysed mechanically at 4 °C by bead beating using a FastPrep™ homogeniser (MP Biomedicals; 5×1 min cycles of shaking at 6 m/s). DNA and protein were removed from the lysed samples by extraction with chloroform (×2) and total RNA purified using RNeasy columns (Qiagen). Illumina TruSeq Stranded Total RNA Library Prep Kit