Computational protocol: A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

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Protocol publication

[…] Cells grown to mid-exponential phase in YPD medium were incubated with or without cantharidin (250 µM) for 1 hour, harvested by centrifugation. Isolation of RNA and hybridization to the tiling arrays was performed as described , except that actinomycin D was added in a final concentration of 6 µg/mL during cDNA synthesis to prevent antisense artifacts . Two independent replicates were used for the analysis. Hybridization to Affymetrix Tiling Arrays using the GeneChip Fluidics Station 450 (Affymetrix) was followed by the extraction of intensity values for the probes using the GeneChip Operating Software (Affymetrix). Acquisition and quantification of array images were performed using the Affymetrix tiling analysis software (http://www.affymetrix.com/support/developer/downloads/TilingArrayTools/index.affx). The resulting .BAR files containing probe position and intensities were further analyzed by aligning the probes that match the position of the S. cerevisiae Genome Database list of defined ORFs (http://downloads.yeastgenome.org/chromosomal_feature/saccharomyces_cerevisiae.gff). The log2 of signal intensity of each ORF was defined as the average across the probes associated with the ORF. Quantile normalized datasets were clustered with a correlation similarity metric and the average linkage method using Cluster 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). The cluster was visualized using TreeView software (http://jtreeview.sourceforge.net/). The significance for differential expression was set as log2 (drug/DMSO) >1 and <−1, P-value <0.05 as determined by Student's t test. Significantly up- and downregulated transcripts were further tested for Gene Ontology (GO) biological process enrichment using FunSpec (http://funspec.med.utoronto.ca/) with P-value cutoff of 0.01 and multiple testing correction (Bonferroni) (; ). The probability was calculated using a test employing hypergeometric distribution (see below). To detect cantharidin-specific genes the genes involved in ESR were eliminated from the gene-set. […]

Pipeline specifications

Software tools TreeView, JTreeView, FunSpec
Application Phylogenetics
Organisms Saccharomyces cerevisiae
Chemicals Cantharidin, Glycerophospholipids