Messenger RNA (mRNA) was isolated and hybridized to Agilent yeast microarrays as described in (46 (link)). Briefly, 5-ml cultures were collected on filters and snap frozen in liquid nitrogen. Total RNA was extracted using the Qiagen RNeasy Mini kit, including the additional DNase I digestion step. Chromosomal RNA (cRNA) for microarray hybridization was synthesized following the standard protocol of the Agilent Low RNA Input Linear Amplification kit (Agilent Technologies). cRNA was extracted using the Qiagen RNeasy Mini kit and hybridized to Agilent Yeast Gene Expression Microarray (8 × 15K G4813A) slides and scanned at 5-μm resolution. Data were extracted using Agilent Feature Extraction software version 9.5 with Linear Lowess dye normalization and no background subtraction and were submitted to the Princeton University Microarray database for storage and analysis.
For estradiol induction experiments, time course fold change in transcript levels was fit to a Hill plot by optimization of n, f0, K and Vmax for each gene for the equation f(t) = f0 + Vmax·tn/(Kn + tn). Delay times were determined by extrapolation of the derivative of this function at f(t) = Vmax/2 to the x-axis intercept.
For estradiol induction experiments, time course fold change in transcript levels was fit to a Hill plot by optimization of n, f0, K and Vmax for each gene for the equation f(t) = f0 + Vmax·tn/(Kn + tn). Delay times were determined by extrapolation of the derivative of this function at f(t) = Vmax/2 to the x-axis intercept.
