Affymetrix GeneChip Yeast Genome S98 [YG_S98]

Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design

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A three-factor design was applied to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae.

GROWTH_RATE

NUTRIENT_LIMITATION

OXYGEN_AVAILABILITY

Transcription profiling of yeast grown in a three-factor design to study the relationship between specific growth rate and genome-wide gene expression

A three-factor design was applied to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae.


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MTAB-2790

The reference laboratory strain S. cerevisiae CEN.PK113-7D (MATa) was grown in well controlled 2 liter jacketed chemostats (Braun Biotech and Applikon) with a constant working volume of 1.0 liter. Cultivations were carried out (in triplicates) in aerobic/anaerobic and carbon/nitrogen limited conditions, at 30°C with a stirrer speed of 800 rpm, pH of 5.0 and dilution rates of 0.03, 0.1 and 0.2 h^-1. Aerobic conditions were maintained by sparging the cultures with air (1.0 L*min^-1). Anaerobic conditions were maintained by sparging the medium reservoir and the fermentor with pure nitrogen gas (0.5 L*min^-1). The bioreactors were fitted with cooled condensers (2 – 4°C) and the off-gas was led to a gas analyzer to measure the content of CO2 and O2. Steady-state was reached when at least five residence times had passed since starting the continuous cultivation and carbon dioxide evolution, dry weight measurements, and HPLC measurements of extracellular metabolites were constant. The experimental work was divided into two efforts. Aerobic cultivations were carried out in the laboratory of Jens Nielsen. Anaerobic cultivations were carried out in the laboratory of Jack T. Pronk. Triplicate cultivations were carried out for each set of conditions to reduce bias that might unexpectedly arise and to account for biological variance. For N-limited cultivations, residual glucose concentration in the chemostat was targeted to 17 ± 2 g*L^-1. This was to sustain glucose repression at the same level in all cultivations. The mineral medium composition for the N-limited cultivations was (amounts per liter): (NH4)2SO4 1 g, KH2PO4 3 g, K2SO4 5.3 g, MgSO4•7H2O 0.5 g, Trace Metal Solution 1 mL, antifoaming agent 0.05 mL and vitamin solution 1 mL. The mineral medium composition for the C-limited cultivations was (amounts per liter): (NH4)2SO4 5 g, KH2PO4 3 g, MgSO4•7H2O 0.5 g, Trace Metal Solution 1 mL, antifoaming agent 0.05 mL and vitamin solution 1 mL. The inlet glucose concentration was ca. 11 and 25 g*L^-1 for aerobic and anaerobic experiments, respectively. Moreover, anaerobic cultivation medium was supplemented with Tween 80/ergosterol solution (1.25 mL/L).

P-MTAB-2791

Samples for RNA isolation from aerobic cultivations were taken by rapidly sampling 20 mL of culture into a tube with 35–40 mL of crushed ice in order to decrease the sample temperature to below 2°C in less than 10 seconds. Cells were then centrifuged (4500 rpm at 0°C for 3 minutes), instantly frozen in liquid nitrogen and stored at - 80°C until further use. Sampling for RNA isolations from anaerobic cultivations was performed as described by Piper et al. (Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 2002).

P-MTAB-2792

Total RNA was extracted using FastRNA Pro RED kit (QBiogene, Inc, USA) according to manufacturer's instructions after partially thawing the samples on ice. RNA sample integrity and quality was assessed prior to hybridization with an Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kit.

P-MTAB-2793

cDNA synthesis and labeling, as well as array hybridization to Affymetrix Yeast Genome S98 arrays, were performed as described in the Affymetrix users' manual. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneArray Scanner.

Transcription profiling of yeast grown in a three-factor design to study the relationship between specific growth rate and genome-wide gene expression

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