Osmotic Stress: Cultures were grown to mid-log densities (between 5x10^7 and 1x10^8 cells/mL) at 30C and treated with KCl to a final concentration of 0.2M, 0.4M, or 0.8M. Samples were harvested at different time intervals (10, 20, 40, 80 minutes after treatment). After growth, 150ml of each culture was transferred to each of 100ml BMW (CTRL - defined in the Growth protocol) and 100ml BMW+KCl (EXP). Both CTRL and EXP media were pre-warmed for 40-60 minutes in the shaker prior to the experiment. EXP media was either BMW + 0.5M KCl, 1M KCl, or 2M KCl, yielding a final concentration of 0.2M KCl, 0.4M KCl, and 0.8M KCl, respectively, upon addition of the culture. In each case, CTRL media was added first, followed by the EXP media, whereupon the shaker was immediately activated to 180 rpm and the timer started simultaneously. Samples (20ml) were collected from the CTRL media + culture immediately upon activation of the shaker (T=0), then at T=10, 20, 40, and 80 minutes from the EXP media + culture.
Growth protocol
All species were grown in the following rich medium chosen to minimize cross-species variation in growth (termed BMW): yeast extract (1.5%), peptone (1%), dextrose (2%), SC amino acid mix (Sunrise Science) 2 g/L, adenine 100 mg/L, tryptophan 100 mg/L, uracil 100 mg/L (Thompson D.A., Roy S., et al., 2012). For each strain, cells were plated onto BMW plates from frozen glycerol stocks. After 2 days, cells were taken from plates and re-suspended into liquid BMW and grown overnight. Approximately 100-1500ul quantities (depending on the species growth rate and timing constraints for the day’s experiments) of the overnight cultures were used to inoculate pre-warmed, 350 ml BMW cultures in 2L Erlenmeyer flasks in New Brunswick Scientific water bath model C76 shakers. All strains were grown at 180 rpm at 30 °C except for S. castellii, which was grown at 25 °C
Extracted molecule
total RNA
Extraction protocol
Qiagen Yeast RNA extraction
Label
Cy5
Label protocol
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
Osmotic Stress: Cultures were grown to mid-log densities (between 5x10^7 and 1x10^8 cells/mL) at 30C and treated with KCl to a final concentration of 0.2M, 0.4M, or 0.8M. Samples were harvested at different time intervals (10, 20, 40, 80 minutes after treatment). After growth, 150ml of each culture was transferred to each of 100ml BMW (CTRL - defined in the Growth protocol) and 100ml BMW+KCl (EXP). Both CTRL and EXP media were pre-warmed for 40-60 minutes in the shaker prior to the experiment. EXP media was either BMW + 0.5M KCl, 1M KCl, or 2M KCl, yielding a final concentration of 0.2M KCl, 0.4M KCl, and 0.8M KCl, respectively, upon addition of the culture. In each case, CTRL media was added first, followed by the EXP media, whereupon the shaker was immediately activated to 180 rpm and the timer started simultaneously. Samples (20ml) were collected from the CTRL media + culture immediately upon activation of the shaker (T=0), then at T=10, 20, 40, and 80 minutes from the EXP media + culture.
Growth protocol
All species were grown in the following rich medium chosen to minimize cross-species variation in growth (termed BMW): yeast extract (1.5%), peptone (1%), dextrose (2%), SC amino acid mix (Sunrise Science) 2 g/L, adenine 100 mg/L, tryptophan 100 mg/L, uracil 100 mg/L (Thompson D.A., Roy S., et al., 2012). For each strain, cells were plated onto BMW plates from frozen glycerol stocks. After 2 days, cells were taken from plates and re-suspended into liquid BMW and grown overnight. Approximately 100-1500ul quantities (depending on the species growth rate and timing constraints for the day’s experiments) of the overnight cultures were used to inoculate pre-warmed, 350 ml BMW cultures in 2L Erlenmeyer flasks in New Brunswick Scientific water bath model C76 shakers. All strains were grown at 180 rpm at 30 °C except for S. castellii, which was grown at 25 °C
Extracted molecule
total RNA
Extraction protocol
Qiagen Yeast RNA extraction
Label
Cy3
Label protocol
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
Hybridization protocol
Standard Agilent protocols for Agilent 8x15K and 4x44K Oligo Microarrays
Scan protocol
Arrays were scanned using an Agilent scanner and analyzed with Agilent’s feature extraction software version 10.5.1.1
Description
0.4MKCl-10_252035410013_2_3
Data processing
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction and LOWESS normalization. Reported expression values for each gene are median log2 ratios across all probes
