Total RNA was isolated according to manufacturer’s instructions using the mirVana™ miRNA Isolation kit (Ambion, Austin, TX). Total RNAs were DNase treated (Turbo DNA-free™, Ambion, Austin, TX), quantified on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Rockford, IL), assessed for quality on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and amplified/biotin-labeled using the Illumina® TotalPrep™ RNA Amplification kit (Ambion, Austin, TX). Aliquots of labeled cRNA were sent to the Yale Center for Genome Analysis (West Haven, CT) where they were hybridized to Illumina® MouseRef-8 v2 Expression BeadChips (Illumina, Inc., San Diego, CA) according to manufacturer protocols. As each beadchip contains 8 independent arrays, samples were hybridized to beadchips in a group counter-balanced format to minimize batch effects. Each array was hybridized with material obtained from a single animal. Each expression array contains approximately 25,600 transcripts representing over 19,100 unique genes. Transcript abundance was measured by fluorescent intensity after scanning. Microarray data have been submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession no GSE42789.
