Genechip fluidics station 450 instrument

Total RNA was isolated from canine J3T-1, J3T-2, J3T-1shA, and J3T-2A cells using RNeasy kit (Qiagen, Santa Clarita, CA, USA) and samples were analyzed using a GeneChip Canine Genome 2.0 Array (Affymetrix, Santa Clara, CA, USA). The microarray analyses were performed by Takara Bio Inc. Briefly, biotinylated cRNA was synthesized from 250 ng total RNA using the GeneChip 3′ IVT PLUS Reagent Kit (Affymetrix), according to the manufacturer’s instructions. Biotinylated cRNA yields were checked with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Scotts Valley, CA, USA). Following fragmentation, 15 μg of cRNA was hybridized for 16 h at 45 °C on a GeneChip Canine Genome 2.0 Array, which were then washed and stained using the GeneChip Fluidics Station 450 instrument (Affymetrix). Arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix). Data from the single-array analyses were calculated using the Microarray Suite version 5.0 (MAS 5.0; Affymetrix) with the default settings and global scaling as the normalization methods. The trimmed mean target intensity of each array was arbitrarily set to 100. A significant change in gene expression was defined as an absolute fold change in expression of ≥2.0 with a P value of < 0.05 compared with appropriate controls. The microarray data were deposited in the Gene Expression Omnibus (GEO) under accession number GSE138374.