Genome wide snowball array

For expression analysis a subset of animals was selected aiming for a balanced design in terms of gender, batch and ancestry. Accordingly, twelve castrated males and twelve females per coping group were selected for subsequent analyses, producing 48 individual samples per sampling time point. The PBMCs were isolated from 5 mL blood by centrifugation on a Histopaque density gradient (Sigma-Aldrich, Taufkirchen, Germany), then stored at -80°C. Total RNA was isolated using Qiazol reagent per manufacturer’s directions (Qiagen, Hilden, Germany). Quantification and purification were performed as previously described [24 (link)]. All RNA was stored at -80°C until downstream analyses were performed. For the microarray experiments, individual samples (n = 192) were hybridized on genome-wide snowball arrays (Affymetrix, Santa Clara, CA, USA), a platform invented for genome-wide analyses of the pig transcriptome [25 (link)]. Processing was performed as previously described [24 (link)]. Raw data have been deposited in a MIAME-compliant database, the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) (accession numbers: GSE55418).

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).

Total RNA was isolated using TRI Reagent per manufacturer’s directions (Sigma-Aldrich, Taufkirchen, Germany), then treated with DNase and purified with the column-based NucleoSpin RNA II-Kit (Macherey-Nagel, Düren, Germany). RNA integrity was determined by visualization on a 1% agarose gel containing ethidium bromide and the concentration was measured using the NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). DNA contamination was assessed by PCR amplification of the porcine RPL32 gene (forward primer: 5’-AGCCCAAGATCGTCAAAAAG-3′; reverse primer: 5’-TGTTGCTCCCATAACCAATG-3′). All RNA samples were stored at − 80 °C.
Each RNA sample was transcribed to DNA using the Ambion WT Expression Kit (Ambion, Austin, TX, USA). The DNA preparations were fragmented and labelled with the WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA). DNA preparations were hybridized on genome-wide snowball arrays (Affymetrix), which were invented for genome-wide analysis of the pig transcriptome [22 (link)]. Raw data was generated with Affymetrix GCOS 1.1.1 software and deposited in a MIAME-compliant database [23 (link)], the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo; accession number: GSE94448).