Yeast strains used were L51 (MATa, ura3-52, leu2-3, 112, his4-519, ade1-100, trp1::HisG, hap1::LEU2). L51 was used for studies of oxygen regulation. To avoid variations from the differences accumulated after many generations of growth of strains, we transformed the L51 strain with the HAP1 gene deleted for studies of Hap1 function. Hap1 protein was expressed in L51 cells by transforming an ARS-CEN plasmid bearing the complete HAP1 genomic sequence. For comparison with cells without Hap1 expressed, an empty vector was transformed into L51 cells. The use of Hap1 expression plasmid generated much more reproducible results than the use of different strains. Yeast cells with or without Hap1 expressed grew at similar rates under both anaerobic and aerobic conditions. The UAS1/CYC1-lacZ reporter plasmid was transformed into yeast cells to confirm the expression of Hap1 and the oxygen levels. Cells were grown in yeast synthetic complete media. Co2+-induced cells were grown in the presence of 400 microM cobalt chloride for 6 hours.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from yeast cells exactly as previously described in [Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current protocols in molecular biology: John Wiley & Sons, Inc.; 2000.]. RNA samples were prepared from L51 yeast cells bearing the empty expression plasmid in the presence of 400 microM cobalt chloride for 6 hours. The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data.
Label
biotin
Label protocol
The synthesis of cDNA and biotin-labeled cRNA were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000).
Hybridization protocol
The yeast Saccharomyces cerevisiae genome 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Specifically, the Affymetrix GeneChip Hybridization Oven 640 and the next generation GeneChip Fluidics Station 450 were used for hybridization and chip processing. Chip scanning was performed by using the GeneChip scanner 3000. Initial data acquisition, analysis was performed by using the Affymetrix Microarray suite. By using GCOS1.2 with the advanced PLIER (probe logarithmic intensity error) algorithm, we calculated and examined the parameters reflecting the image quality of the arrays. Arrays with a high background level in any region were discarded and replaced. The average noise or background level was limited to less than 5%. The average intensity for those genes judged to be present was at least 10-fold higher than those judged to be absent. Also, arrays that deviated considerably in the percentage of present and absent genes from the majority of the arrays were replaced. Arrays with a beta-actin 3’/5’ ratio greater than 2 were replaced.
Scan protocol
The yeast Saccharomyces cerevisiae genome 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Specifically, the Affymetrix GeneChip Hybridization Oven 640 and the next generation GeneChip Fluidics Station 450 were used for hybridization and chip processing. Chip scanning was performed by using the GeneChip scanner 3000. Initial data acquisition, analysis was performed by using the Affymetrix Microarray suite. By using GCOS1.2 with the advanced PLIER (probe logarithmic intensity error) algorithm, we calculated and examined the parameters reflecting the image quality of the arrays. Arrays with a high background level in any region were discarded and replaced. The average noise or background level was limited to less than 5%. The average intensity for those genes judged to be present was at least 10-fold higher than those judged to be absent. Also, arrays that deviated considerably in the percentage of present and absent genes from the majority of the arrays were replaced. Arrays with a beta-actin 3’/5’ ratio greater than 2 were replaced.
Description
L51 yeast cells bearing the empty expression plasmid in the presence of 400 microM cobalt chloride for 6 hours. Cells were grown in yeast synthetic complete media. For RNA preparations, yeast cells were inoculated so that the optical density of yeast cells was in the range of 0.8-1.0 immediately before the collection of cells.
Data processing
For each microarray, we converted the .DAT image files into .CEL files using the Affymetrix GCOS software. These raw .CEL files were further processed into expression values using the RMA express software by Bolstad. This software uses the robust multiarray average method by Irrizary et al. which involves a background correction and a quantile-based normalization scheme.
