An industrial wine strain of S. cerevisiae, Vin13 (Anchor Yeast, South Africa), was used. The dried yeast was resuspended (Marks et al, 2003) and inoculated into 1 L filter sterilized Riesling grape juice harvested from the Okanagan Valley in British Columbia, Canada in the 2000 growing season. The final cell count was 4 x 106 /ml. The grape juice contained 214 g/L sugar (equimolar amounts of glucose and fructose), 70 mg/ L ammonia, and 140 mg/L free alpha-amino nitrogen (Marks, 2003 et al, 2003). Incubation was at 20 °C
Extracted molecule
total RNA
Extraction protocol
extraction of total RNA was done according to (Causton et al, Mol. Biol. Cell 12, 323–337,2001). Double-stranded cDNA was purified using phenol/chloroform/isoamyl alcohol (25:24:1) saturated with 10 mM Tris–HCl, pH 8.0, 1 mM EDTA (Ambion, Austin, TX, USA) and a Phase Lock Gel prepared according to the manufacturer's instructions (Eppendorf, Hamburg, Germany)
Label
biotin
Label protocol
Following ethanol precipitation, cRNA was prepared by in vitro transcription of the cDNA using the Enzo BioArray™ HighYield™ RNA Transcript Labeling kit (Farmingdale, NY, USA). The cRNA was purified with RNeasy spin columns (Qiagen, Mississauga, ON, Canada), followed by ethanol precipitation. Then, 20 μg of cRNA (0.5 μg μl-1) was fragmented by incubating at 94°C for 35 min in fragmentation buffer (40 mM Tris–acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Further details of procedures can be found in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
20 μg of cRNA (0.5 μg μl-1) was fragmented by incubating at 94°C for 35 min in fragmentation buffer (40 mM Tris–acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate)
Scan protocol
as described by the manufacturer (Eukaryotic Arrays GeneChip Expression Analysis and Technical Manual, Affymetrix, Santa Clara, CA), using an Agilent G2500A GeneArray Scanner
Description
yeast cells harvested after 12 hours
Data processing
RMA applied to the whole series (as implemented in the 'affy' package in Bioconductor)
