MinION sequencing; RNA-seq of 8-week-old ram testes exposed gestationally to chemical mixture
male lambs from ewes exposed to conventionally fertilised pastures (n=7, Control), and ewes from BTP (n=17, biosolid) were weighed before euthanasia by intravenous barbiturate overdose. Tissues were collected and stored at -80 C until RNA extraction. RNA was extracted and purified from approximately 25-30 mg of homogenised tissue with an RNeasy Kit (Qiagen – 74104) supplementing the RLT buffer with 1% (v/v) ß-mercaptoethanol (Sigma-Aldrich – M3148). The purity and concentration of RNA was assessed spectrophotometrically using a NanoDrop (Thermo Scientific, model ND-1000).  For each sample, 100 ng of extracted RNA was converted to cDNA using RNaseOUT (Invitrogen – 10777019), Maxima H Minus Reverse Transcriptase (Thermo Scientific – 15259496), primers from a Direct cDNA Sequencing Kit (Oxford Nanopore – SQK DCS109), RNase Cocktail (Invitrogen – AM2286), LongAmp Taq Master Mix (New England Biolabs – M0287), NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs – E7546), and AMPure XP magnetic beads (Beckman Coulter – A63881). cDNA was then assessed spectrophotometrically, as above, for use in Nanopore sequencing.