Gc3000 g7 scanner

Microarray analysis was performed with the GeneChip Mouse Gene 2.0 ST array (Thermo Fisher Scientific Inc, CA, USA) using targets derived from total RNA isolated from NAc as described above. cDNA for array hybridization was synthesized from 150 ng of total RNA from each single mouse using a GeneChip WT Plus Kit (Thermo Fisher Scientific Inc, CA, USA) according to the manufacturer’s specifications. Briefly, total RNA was used to synthesize double-stranded cDNA, which was then used as a template for anti-sense RNA (cRNA) synthesis. This cRNA was in turn used as a template for a second round of single-stranded cDNA (sense strand cDNA, ss-cDNA) synthesis, and the resultant DNA-RNA hybrids were then treated with RNase H to degrade RNA. The ss-cDNA was then fragmented and biotinylated using GeneChip WT Terminal Labeling Kit (Thermo Fisher Scientific Inc, CA, USA) according to the manufacturer’s specifications. Fragmented and labeled ss-cDNA targets were hybridized with the arrays at 45°C for 16 hours. The hybridized arrays were then washed and stained according to manufacturer specifications, and arrays were scanned on an Affymetrix GC3000 G7 Scanner. Data were extracted and processed from scans using Affymetrix Command Console v. 3.1.1.1229. cDNA synthesis, fragmentation, labeling, array hybridization, staining, and scanning were performed by the University of Chicago Genomics Core.