normalization data transformation protocol
The average intensity of the Cy3 and Cy5 fluorescence at each spot was then extracted using GenePix 5.1 software. Lowess and quantile normalization were performed using the MATLAB bioinformatics toolbox before further analysis.
array scanning protocol
Microarrays were washed and scanned immediately using an Axon 4000B scanner
nucleic acid hybridization to array protocol
300 ng Cy3-labeled and 300 ng Cy5-labeled cDNA was competitively hybridized to Agilent 8_15K S. cerevisiae two-color expression microarrays (G2509F) in Agilent hybridization buffer for 17 hours at 60 degrees C.
nucleic acid labeling protocol
Purified cDNA samples were then labeled with NHS-ester Cy3 or Cy5 (GE Biosciences).
nucleic acid extraction protocol
Cell cultures were added directly into cold methanol (~ -50 ¡C) with volume ratio of 2:3, and incubated in an ethanol-dry ice bath for at least 20 minutes. Cells were collected by centrifugation and quickly washed with ice-cold water to remove alcohol, and resuspended in RNAlater solution (Ambion). For each sample, 5_107 cells were used to isolate total RNA with the RNase Mini kit (Qiangen), and RNA integrity was analyzed on an agarose gel or with an Agilent bioanalyzer. cDNA was synthesized from 10 _g total RNA with 1:1 ratio of random 10-mers and oligo-dT primers (Operon), and a 2:3 ratio of amino allyl-UTP:dTTP (Sigma), using the Superscript III reverse transcription system (Invitrogen). cDNA was purified with a PCR purification kit (Qiagen) after hydrolyzing RNA.
Yeast strains were grown usinglow fluorescemce medium at 30 degrees C with shaking and cell samples were collected at early logarithmic phase (OD600 0.15-0.2).
To inhibit PKA activity, yeast cells at early logarithmic phase were treated with 3 uM of PKA inhibitor 1-NM-PP1 for indicated times.