Ht 12 v4.0 beadchips

Total RNA extracted using Trizol reagent (Invitrogen) and verified for integrity and purity, was labeled using Illumina TotalPrep RNA amplification kit (Ambion, Austin, TX) as per manufacturer's instructions. The cRNA generated was hybridized to Illumina HT-12 v4.0 BeadChips at 58°C for 18 h. The BeadChips were then washed, stained, and scanned using iScan (Illumina). Data files were quantified in GenomeStudio v2011.1 (Illumina), quantile normalized using the lumi package of Bioconductor [33] (link), and filtered using varFilter to retain the 10% most variable probes. Data for multiple probes targeting the same gene were collapsed to their median value and annotated with gene names using GenePattern [34] (link). The collapsed data were analyzed using the linear models for microarray analysis (LIMMA) package [35] (link) to identify differentially expressed genes. To understand the relationship among differentially expressed genes further, we performed a network-based analysis using the Reactome Functional Interaction (FI) Network plugin for Cytoscape [36] (link). The gene expression profiling data can be accessed from the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo. Accessed 2014 May 1), accession number GSE54608.