Microarray design and analysis
Microarray experiments were performed at the VIB Nucleomics Core (www.nucleomics.be). Before labeling, RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 1 μg of total RNA spiked with 10 viral polyA transcript controls (Agilent, Santa Clara, CA, USA) was converted to double stranded cDNA in a reverse transcription reaction. Subsequently, the samples were converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) or Cyanine 5-CTP (Cy5) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent, Santa Clara, CA, USA). A mixture of purified and labeled cRNA (Cy3 label: 5 pmol; Cy5 label: 3.5 pmol) was hybridized on Agilent Yeastv2 arrays followed by washing, according to the manufacturer's procedures. To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner and probe signals were quantified using Agilent's Feature Extractor software (version 10.5.1.1).
In total four different comparisons were made. Each comparison was done in duplo by a color flip. The different comparisons are shown in Figure 1A. Gene expressions of strains transformed with wtDFNA5 were compared to strains transformed with mutDFNA5, both in mid-exponential phase (comparison 1a) and at the post-diauxic shift (comparison 1b). Additionally, comparisons were made between the mid-exponential and the post-diauxic shift of either wtDFNA5-(comparison 2b) or mutDFNA5-(comparison 2a) transformed cells.
Analysis of the microarray data was performed using the R package LIMMA (http://www.bioconductor.org) (Gentleman et al., 2004). Fold changes were computed using raw Cy3 and Cy5 intensities values provided by Agilent's Feature Extractor software (version 10.5.1.1) and loess normalization and background correction were performed to determine the log2-ratios per array. Differential expression was assessed via the moderated t-statistic, described in (Smyth, 2004). To control the false discovery rate, multiple testing correction was performed. This generated four differentially expressed gene lists. All the (raw) data files have been completely uploaded to the gene expression omnibus and have been stored under accession number GSE70169.