Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8. Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C. An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation. Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C. For more information on the unit and software, contact the corresponding author. A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process. Samples were collected on a volume basis and were in the same ratio as collected at T1. Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry). Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask. Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration. Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092). RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio. Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E1_T3_R3, Dry.
