Harvested as explained in the mechanical disruption protocol of the RNeasy Mini Kit (50), Qiagen.
Growth protocol
Minimal medium, glucose as carbon source, 30°C, 300 rpm, exponential growth in batch.
Extracted molecule
total RNA
Extraction protocol
Extraction as explained in the mechanical disruption protocol of the RNeasy Mini Kit (50), Qiagen.
Label
biotin
Label protocol
Total RNA samples were reverse-transcribed with One-Cycle cDNA Synthesis Kit (Affymetrix Inc., P/N 900431, Santa Clara, CA). The double-stranded cDNA was purified using the Sample Cleanup Module (Affymetrix Inc.,P/N 900371). The purified double-stranded cDNAs were in vitro transcribed with biotin labeled nucleotides using the IVT Labeling Kit (Affymetrix Inc., P/N 900449).
Hybridization protocol
Biotin-labeled cRNA samples were fragmented randomly to 35-200 bp at 94°C in fragmentation buffer (Affymetrix Inc., P/N 900371) and suspended in 100ml of hybridization mix (Affymetrix Inc., P/N 900720) containing a hybridization control and control oligonucleotide B2 (Affymetrix Inc., P/N 900454). Samples were hybridized to GeneChip Yeast Genome 2.0 arrays for 16h at 45°C.
Scan protocol
An Affymetrix GeneChip Scanner 3000 (Affymetrix Inc.) was used to determine the fluorescent intensity emitted by the labeled target.
Description
Gene expression of wild-type FY4 during exponential growth in minimal media with glucose as carbon source.
Data processing
Affymetrix CEL files were processed using R (version 2.8.0) (R Development Core Team, 2008) and the Bioconductor affy package. Probe intensities were normalized for background by using the robust multiarray average method (RMA), using only perfect match (PM) probes. Normalization was performed using the qsplines algorithm. Gene expression values were calculated from the PM probes using the expression index calculation method (Li and Wong, 2001). All the normalization steps were done for pairwise conditions, i.e., mutant vs. wild-type.
