Transcriptomic signatures of the porcine oviduct epithelial cells responding to an administration of cortisol and two selective glucocorticoid receptor (GR) antagonists; AL 082D06 and Cort108297 in an air-liquid-interface culture system
Transcriptomic signatures of the porcine oviduct epithelial cells responding to an administration of cortisol and two selective glucocorticoid receptor (GR) antagonists; AL 082D06 and Cort108297 in an air-liquid-interface culture system
The air-liquid interface culture of porcine oviduct epithelium has been prepared as previously described (Chen et.al. 2013; Biol Reprod; doi:10.1095/biolreprod.113.108829). Cell cultures from 6 biological replicates (individual donor pigs) were treated from the basolateral side with 1) basal media control; 2) 250 nM cortisol; 3) 250 nM cortisol + 0.25µM glucocorticoid receptor (GR) antagonists AL 082D06; 4) 250 nM cortisol + 2.5µM AL 082D06; 5) 250 nM cortisol + 25µM AL 082D06; 6) 250 nM cortisol + 0.025µM glucocorticoid receptor (GR) antagonists Cort108297; 7) 250 nM cortisol + 0.25µM Cort108297; 8) 250 nM cortisol + 2.5µM Cort108297. Cells were harvested for RNA-seq analysis after 12-hours of the treatments. The 12-well ALI-POEC cultures were washed with pre-warmed PBS before RNA isolation using a commercial kit (NucleoSpin RNA, Macherey-Nagel). RNA quantification was performed by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies). RNA integrity values of all samples were higher than 8.9 on average. Total RNA of one microgram (RIN > 8) was used for library preparation using an Illumina Stranded mRNA Prep, Ligation kit with 10 PCR-cycles of amplification according to the manufacture's recommendation (Illumina). The libraries were quality checked for fragment length distribution on Agilent 2100 Bionalyzer and normalized to an equal concentration of 10nM prior to pooling.