Agilent feature extraction software v 9.5.1

Microarray analyses were conducted as described previously [44 (link)]. Maize internode sections were ground under liquid nitrogen and total RNA extracted with the TRIZOL reagent (Invitrogen, Sydney) according to the manufacturer’s instructions. Polyadenylated mRNA was purified using the Illustra mRNA Purification Kit (GE Biosciences). Sample quality and RNA concentration were assessed using an Agilent Bioanalyzer. The mRNA was reverse-transcribed into double-stranded cDNA, which was labelled with Cy3 or Cy5 fluorescent dye, using the Agilent Low RNA Linear Amp kit. Biological replicates were labelled alternately using Cy3 or Cy5 to guard against dye-bias. The cDNA was hybridized to Agilent 4 × 44 k maize gene microarrays [70 (link)] and the microarrays were washed according to Agilent standard protocols. The microarray chips were scanned with an Agilent G2505B DNA Microarray Scanner at two laser power settings (100% and 10%). The images were inspected visually for image artefacts, and feature intensities were extracted, filtered and normalized with Agilent Feature Extraction Software (v 9.5.1). The data were normalized using quantile normalization (BOLSTAD http://www.ncbi.nlm.nih.gov/pubmed/12538238). The normalized data were used to compare expression levels of genes related to cell wall compositions.

Detailed procedures for ssDNA labeling, microarray hybridization and data analysis were described previously [39 (link)]. Briefly, cell cultures were grown to an OD₆₀₀ of 0.25~0.3 in YPD medium, followed by G1 arrest with 200 nM α-factor. Pronase (0.02 mg/mL) was used to synchronously release cells into S phase in the presence of 200 mM hydroxyurea (HU). G1 control and S phase samples were collected prior to cell cycle release and after 1 h treatment of HU, respectively. Three-hundred ml of cells from each sample were collected and spheroplasted in agarose plugs for ssDNA labeling. Differentially labeled G1 (Cy5-dUTP) and S phase (Cy3-dUTP) DNA were co-hybridized onto Agilent Yeast Whole Genome ChIP-on-chip 4 × 44K (G4493A) microarrays and the data were extracted by the Agilent Feature Extraction Software (v9.5.1). The relative quantity of ssDNA at a given genomic locus was calculated as the ratio of the fluorescent signal from the S phase sample to that of the G1 control, followed by Loess-smoothing over a 6-kb window at a step size of 250 bp.