Gis2 mutant versus wild-type cells

To measure the relative changes of gene expression upon Gis2p depletion, 50 ml of delta Gis2 cells or the respective wild-type strain BY4741 were grown in YPD at 30 degrees celsius to an OD600 of 0.5- 0.6. Cells were harvested by centrifugation and washed twice with 5ml of bidistilled water (ddH2O). RNA was isolated by hot phenol extraction for microarray analysis. 20 ug of total RNA derived from wild-type and knock-out cells were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized onto yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices) before uploading into Stanford Microarray Database. Biological Replicate