Pulmonary CD11 cells separated using magnetic beads (miltenyi). Total RNA from CD11c cells extracted using mirVana kit (Life Technologies) according to manufacturer's protocol. Quality checked using agilent Bioanalyzer.
DNA microarray sample processing. RNA sample preparation for hybridization and the subsequent hybridization to the Illumina beadchips were performed at the University of Iowa DNA Facility using the manufacturer’s recommended protocol. Briefly, 100 nanograms total RNA was converted to amplified Biotin- aRNA using the Epicentre TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Illumina, Inc., San Diego, CA, Cat. #TAN07924) according to the manufacturer’s recommended protocol. The amplified Biotin-aRNA product was purified through a QIAGEN RNeasy MinElute Cleanup column (QIAGEN Cat #74204) according to modifications from Epicentre.
750 ng of this product were mixed with Illumina hybridization buffer, placed onto Illumina MouseRef-8 v2.0 expression beadchip, and incubated at 58º C for 17h, with rocking, in an Illumina Hybridization Oven. Following hybridization, the arrays were washed, blocked, then stained with streptavidin-Cy3 (Amersham/GE Healthcare, Piscataway, NJ) according to the Illumina Whole-Genome Gene Expression Direct Hybridization Assay protocol.
Beadchips were scanned with the Illumina iScan System (ID #N0534) and data were collected using the GenomeStudio software v2011.1.