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Expanded Experimental Methods
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Strains and Media
Expression profiling experiments were performed with the diploid mating type a/α strain BY4743 (Open Biosystems, Huntsville, AL, USA), derived from strains BY4741/BY4742 in the S288C background as a wild-type control. Homozygous diploid knock-out strains (Invitrogen Corporation, Carlsbad, CA, USA) were constructed by the Saccharomyces Gene Deletion Project (http://sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html) based on this parent strain, and all cultures were grown using Synthetic Complete media (SC) with 2% glucose at 30°C.
Culture Growth, RNA Processing, and cDNA Labeling
For each experiment, growth conditions identical to Hughes et al. were employed. Two independent colonies from the wild-type control and knockout strain grown on YPD agar were selected and processed in parallel. 1.0 ml of saturated overnight culture inoculated from each of these single colonies was used to seed 100 ml of SC media pre-warmed to 30°C in a 500-ml Erlenmeyer flask, which was placed in a 30°C shaking incubator at 200 rpm until each culture reached an OD600 of approximately 0.8 - 1.0. Every effort was made to closely match the final optical densities of all cultures to one another. Cells were harvested by centrifugation at 3000 rpm for three minutes at room temperature in a Legend RT™ centrifuge (Kendro Laboratory Products, Asheville, NC, USA), snap-frozen in liquid nitrogen to suspend gene expression, and stored at -20°C prior to RNA extraction. Total RNA from each frozen cell pellet was isolated by hot acid phenol extraction and purified to mRNA using Poly(A)Pure™ kits (Ambion, Austin, TX, USA). Fluorescent labeling was performed by direct incorporation using a CyScribe First-Strand cDNA Labeling Kit (Amersham Biosciences, Piscataway, NJ, USA) in a forward-and-reverse dye labeling scheme.
Array Production and Hybridization
DNA microarrays were spotted with an OmniGrid 100® Microarrayer (Genomic Solutions, Ann Arbor, MI, USA) using the Yeast Genome Oligo Set, Version 1.1 (Qiagen Inc., Valencia, CA, USA) on UltraGAPS amino-silane coated slides (Corning Inc., Corning, NY, USA). After spotting, microarrays were baked at 80°C for 2 hours, cross-linked at 300 mJ in a UV Stratalinker 2400 (Stratagene, La Jolla, CA, USA) to covalently link DNA to the slide, and stored in vacuum desiccators to ensure low humidity and to prevent oxidation of the slide surface. Each single-gene knockout sample was co-hybridized versus the wild-type control in a single two-color microarray experiment. In each experiment, the appropriate Cy3- and Cy5-labeled samples were combined and lyophilized to 5.0 mL volume using a DNA 110 SpeedVac (Savant Instruments, Holbrook, NY, USA) with a medium heat setting. Samples were resuspended in 50.0 mL hybridization solution composed of 5X SSC, 0.1% SDS, 1X Denhardt’s Solution and 25% formamide, and coverslip hybridization was conducted at 42°C for 15 hours. Arrays were then scanned using a ScanArray Lite scanner (PerkinElmer Life and Analytical Sciences, Torrance, CA, USA) at 10.0 μm resolution.
Data Post-processing and Significance Assessment
Scanned images were quantitated using the QuantArray quantitation package (PerkinElmer). Raw quantitated background intensities were smoothed using a 7x7 median filter, separately for the Cy3 and Cy5 channels, and data was corrected for cyanine-dye dependent bias using a Qspline normalization so that the median intensities were constant over all dye channels and all experiments. Resulting log ratios were smoothed in a 9x9 window to further adjust for any spatial bias. The VERA/SAM package (http://db.systemsbiology.net/software/VERAandSAM/) was utilized to estimate multiplicative and additive errors using the method of maximum likelihood, and to apply the resulting error model to associate a log-likelihood statistic λ with each gene, indicating its likelihood of differential expression for each gene in each knockout vs. control experiment. Data was formatted for import into the Cytoscape software package (http://www.cytoscape.org/) to view integration of expression levels with protein-protein and protein-DNA interaction networks.
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