Testes were dissected as described above and transferred to tubes containing 200 μL Tri Reagent Solution (ThermoFisher, Catalog #AM9738). Individual testes were homogenized with a plastic pestle (USA Scientific, Catalog #1415–5390) and vortexed every 2 minutes over 10 minutes and kept at room temperature. 20 μL of 1-Bromo-3-chloropropane (BCP) was added to the samples then vortexed and incubated at room temperature for 5 min before a 15 minute centrifugation at 14,000 RPM. 80 μL of the top clear layer was transferred to a fresh RNase-free 1.5 mL tube. 0.8 μL of 20 μg/mL glycogen (ThermoFisher, Catalog #R0551) and 80 μL of 100% isopropanol were added to each tube and the samples were placed at -20° C overnight. The tubes were centrifuged at 14,000 RPM for 30 min and the supernatant was removed. The pellets were washed with 300 μL of 75% ethanol and allowed to air dry. RNA pellets were resuspended in 7 μL of nuclease-free water (ThermoFisher, Catalog #4387937). RNA was quantified using a NanoDrop 1000 Spectrometer (ThermoFisher). RNA was treated with DNase I to remove genomic DNA (ThermoFisher, Catalog #18068–015). First-strand cDNA was synthesized using SuperScript First-Strand Synthesis System for RT-PCR (ThermoFisher Catalog #11904–018).

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