Cold ethanol

Total RNA was extracted from the cells using the RNAiso reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Briefly, cells (1 × 10⁶ cells/mL) were washed with cold PBS and lysed with RNAiso reagent. Chloroform (Samchun, Seoul, Republic of Korea) was added to the lysate, and the mixture was centrifuged at 12,000× g for 15 min at 4 °C to separate the aqueous and organic phases. The aqueous phase was then transferred to a fresh tube, and RNA was precipitated by adding isopropanol (Duksan, Seoul, Republic of Korea) for 10 min at room temperature (RT). The RNA was centrifugated at 12,000× g for 10 min at 4 °C to precipitate the pellet. Subsequently, the RNA was washed with 75% cold-ethanol (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged at 7500× g for 5 min at 4 °C. The washed RNA pellet was air-dried until the pellet was slightly transparent and dissolved in diethyl pyrocarbonate treated water (Biosesang, Seongnam, Republic of Korea). RNA quality and quantity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA).

Sperm DNA was extracted according to the modified protocol described by Marques et al. [28 (link)]. Briefly, sperm pellets were overlaid with 500 μl of lysis solution (LS) containing 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 50 mM NaCl, 1 mM DTT, and 0.2 mg/ml proteinase K (Fermentas) and incubated overnight at 55 °C. Sperm DNA was isolated by standard phenol chloroform extraction. DNA was precipitated using cold ethanol (Sigma-Aldrich), washed, and solubilized in water. All DNA samples were quantified/qualified using a Nanodrop Spectrophotometer (Invitrogen). Sperm DNA samples were included in the study if the ratio DO260/280 was between 1.8 and 2 and the DNA concentration above 50 ng/μl.