Sperm DNA Extraction from Ejaculates

Extraction of sperm cell DNA from fresh ejaculates was performed as previously described^{40 (link)}. In short, sperm cells were isolated by centrifugation of the fresh (up to 2 days) ejaculate over an isotonic solution (90%) (Sage/Origio, ART-2100; Sage/Origio, ART-1006) using up to 2 mL of the sample. Following a washing step, quantity and quality were assessed using a cell counting chamber (Sigma-Aldrich, BR717805–1EA). Cells were pelleted and lysis was performed by addition of RLT lysis buffer (Qiagen, 79216), Bond-Breaker TCEP solution (Pierce, 77720), and 0.2 mm stainless steel beads (Next Advance, SSB02) on a Disruptor Genie (Scientific Industries, SI-238I). The lysate was processed using reagents and columns from an AllPrep DNA/RNA Mini Kit (Qiagen, 80204). Concentration of the final eluate was assessed employing standard methods. Concentrations ranged from ~0.5–300 ng/μl.