Transcriptome Analysis of Porcine PBMC

Samples obtained at 28 dpn and 63 dpn were used to isolate peripheral blood mononuclear cells (PBMC) from 5 ml of blood by centrifugation on a Histopaque density gradient (Sigma-Aldrich, Taufkirchen, Germany), and then stored at −80°C. Total RNA was isolated using Qiazol reagent per the manufacturer's directions (Qiagen, Hilden, Germany). Quantification and purification were performed as previously described (52 (link)). All RNA samples were stored at −80°C until transcriptome profiling was performed. Individual RNA samples were transcribed to DNA using the Ambion WT expression kit (Ambion, Austin, TX). DNA preparations were fragmented and labeled using the WT terminal labeling kit (Affymetrix, Santa Clara, CA). Subsequently, preparations were hybridized on genome-wide “snowball” arrays (Affymetrix, Santa Clara, CA), a platform invented for genome-wide analyses of the pig transcriptome (23 (link)). Raw data were generated using Affymetrix GCOS 1.1.1 software and were deposited in a MIAME-compliant database (19 (link)), the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo; accession number: GSE66308).