Illumina HiSeq 2500 paired end sequencing; RNA-seq of porcine peripheral blood mononuclear cells treated with lipopolysaccharide and dexamethasone
Peripheral blood mononuclear cells (PBMCs) were isolated 10 ml whole blood samples taken from anterior vena cava punctures and collected into pre-chilled tubes with EDTA. The blood samples were then centrifuged on a Histopaque-1077 density gradient (Sigma-Aldrich, Taufkirchen, Germany) following the manufacturer's instructions to recover the layer of PBMCs. The isolated PBMCs were stored in liquid nitrogen with 90% fetal bovine serum (FBS; PAA, Cölbe, Germany) and 10% DMSO until use. PBMCs were thawed and re-suspended in cell culture medium (RPMI 1640 medium supplemented with 10% FBS, 2 mmol/l L-glutamine (Biochrom, Berlin, Germany), 100 U/ml penicillin (PAA, Cölbe, Germany), and 100 mg/ml streptomycin (PAA, Cölbe, Germany) and adjusted to 6 × 106 cells/ml. PBMCs for each animal were divided into four groups and seeded in 24-well plates at 3 × 106 cells/well, followed by the overnight incubation at 37 °C with 5% CO2. Then, the cells were treated with either 1) vehicle (CON), 2) DEX (5nM in ethanol (≈ 0.002 μg/ml); Dexatat, aniMedica, Senden, Germany), 3) LPS (10 μg/ml in PBS; Escherichia coli O111: B4; Sigma-Aldrich, Taufkirchen, Germany) or 4) combination of LPS+DEX and further incubated for 2 hours at 37 °C with 5% CO2. Afterward, cells were harvested for RNA extraction.  Total RNA was extracted from PBMCs using the TRI reagent (Sigma-Aldrich, Taufkirchen, Germany) and purified by the RNA Clean&Concentrator-25 Kit (Zymo Research, Freiburg, Germany) following the manufacturer's instructions. Genomic DNA was removed by the RNase-Free DNase Set (Qiagen, Hilden, Germany). RNA integrity was assessed by the Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano kit (Agilent Technologies, Waldbronn, Germany). 1 µg of total RNA with RIN > 8 was used for library preparation using a TruSeq Stranded mRNA Kit with 11 cycles of PCR-amplification according to the manufacture's recommendation (Illumina).