NextSeq 500 sequencing; Fetal androgen exposure determines adult male health
Fetal androgen exposure determines adult male health
All animals received a bolus injection of glucose (10 g glucose in 20 ml saline), and 15 minutes post injection sacrifice was achieved via barbiturate overdose. Tissues were recovered immediately and snap frozen prior to storage at -80 degrees celsius until downstream analysis. In each animal, liver sampling occurred from the right posterior lobe, in approximately the same area in all animals.  Mature Scottish Greyface ewes were fed in order to achieve a comparable body condition score (2.75–3) prior to estrous cycle synchronisation. After a synchronised mating (Texel ram) animals were allocated at random to one of two experimental groups – vehicle control or testosterone propionate (TP) exposed. On day 62 and day 82 of gestation, anaesthesia was induced by initial sedation via administration of 10 mg Xylazine i.m ('Rompun', Baylor plc Animal Health Division, UK), followed by 2 mg/kg ketamine (i.v, Keteset, Fort Dodge Animal Health, UK). All downstream procedures were conducted under surgical aseptic conditions. TP was dissolved in vegetable oil (100 mg/ml) and a 200 μl volume injected (20 G Quinke spinal needle, BD Biosciences) via ultrasound guidance into the fetal flank. Control fetuses received 200 μl vegetable oil vehicle alone. This procedure was then repeated at d82 of gestation. Immediately after surgical procedure completion all pregnant ewes were given prophylactic antibiotics (Streptacare, Animalcare Ltd, UK, 1 ml/25 kg) and were then monitored during recovery. RNA was extracted and purified using Qiagen AllPrep kits following manufacturer's instructions. RNA concentrations were determined using a NanoDrop 1000 spectrophotometer with Agilent Bioanalyser analysis utilised for RNA quality control. RNA extracts were stored at -80 degrees celsius until further analysis. 1 microlitre of ERCC spike in controls were added to 500 ng of RNA sample. One if the two ERCC mixes were selected at random for each sample. Libraries were prepared with the Illumina TruSeq Stranded mRNA kit.