Sodium dodecyl sulfate (sds)

A total of 6 L of each water samples from the Mulgol were collected and filtered through 0.45 µm pore-sized Durapore membrane filters (Merck Millipore, Billerica, MA, USA) using a vacuum pump (model DOA-P704-AC, GAST, Benton Harbor, MI, USA) during the field periods. Probably, dissolved extracellular DNA passed through 0.45 µm pore-sized membrane filters^{50 (link)}. These filters were stored in a 50 mL conical tube at −20 °C and taken to the laboratory for further experiments.
For environmental DNA extraction, 20% (w/v) lysozyme (final concentration, Sigma-Aldrich, St. Louis, MO, USA) were directly added to each conical tube after the filters were cut into several pieces. The tubes were then incubated at 37 °C for 30 min. Further, 0.5 mg mL⁻¹ proteinase K (final concentration, Sigma-Aldrich) and 1% sodium dodecyl sulfate (final concentration, Bioneer, Daejeon, Korea) were added to the conical tube, and the tubes were incubated at 55 °C for 2 hrs. Nucleic acids were further purified using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. The concentration of extracted DNA was measured with Quantus fluorometer (Promega, Madison, WI, USA). The final concentrations of extracted DNA ranged from 0.68 to 14.19 ng μL⁻¹.

Genomic DNA (gDNA) was extracted from the rat ovaries via treatment with proteinase K (QIAGEN, Valencia, CA, USA) and phenol/chloroform (Sigma-Aldrich). The ovary tissues of rats were ground in LN2, and the powdered tissue was then digested overnight at 55 °C in digestion buffer [100 mM Tris pH 8.0 (Abelbio, Jungnang, Seoul, Korea) containing 5 mM EDTA (Bioneer, Daedeok, Daejeon, Korea), 0.2% sodium dodecyl sulfate (SDS; Bioneer), 200 mM sodium chloride (Bioneer), and 0.5 mg/ml proteinase K (Dako, Cambridge, U.K.)]. The supernatants containing gDNA were extracted by using phenol/chloroform (1:1, Sigma-Aldrich) and precipitated with isoamyl alcohol (Sigma-Aldrich) and 0.3 M sodium acetate (Bioneer) at − 20 °C overnight. Next, the gDNA pellet was washed with cold 70% ethanol and eluted using a Tris-EDTA buffer. The gDNA for each sample was analyzed using 1% agarose gel electrophoresis.