Wild type parental strain (BY4741) cells grown in glucosamine (15 mM) overnight were diluted 1:50 into fresh YPD supplemented with glucosamine and allowed to grow to a mid-log phase (OD600 1.6-1.8), and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda=260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite. Three replicates labeled #1, #2, and #3 were performed. Keywords = saccharomyces chitin glucosamine
