DRIP-seq control bonobo sperm
DRIP was performed as previously described with minor modifications (Halsz et al., 2017; Bou-Nader et al., 2022). Briefly, SureBeads Protein A Magnetic Beads (BioRad) were pre-blocked with PBS containing 0.5% BSA and 5 mM EDTA and subsequently washed with wash buffer (PBS; 1% Triton X-100; 1 mM EDTA). To pre-immobilize the S9.6 antibody, 100 L pre-blocked beads were incubated with 5 g of S9.6 antibody in binding buffer (50 mM TRIS; 0.14 M NaCl; 5 mM EDTA; 1% Triton X-100) at 4 C for 2 h with rotation. DNA was isolated from sperm head pellets using phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol and resuspended in TE buffer. The digested DNA was added to the beads and incubated overnight at 4 C. Bound beads were recovered and washed three times with wash buffer (PBS; 1% Triton X-100; 1 mM EDTA). Precipitates were eluted in elution buffer (10 mM Tris pH 8; 1 mM EDTA; 1% SDS) and 5 l proteinase K (10 mg/ml) in 100 l for 15 min at 65 C. DNA was purified using a QIAquick PCR purification Kit (QIAGEN).