GSM4970016: H3K27me3_Sperm_WT; Mus musculus; OTHER
Sperm were collected using the "swim-up" method Mouse sperm H2Aub ChIP-seq was performed as described previously with some modifications. Briefly, ~2 million mouse spermatozoa (PWK) collected by the swim-up method were treated with 50 mM DTT (Sigma-Aldrich) for 2 hrs at room temperature and quenched by adding N-ethylmaleimide (NEM, Sigma-Aldrich) and incubating for additional 30 min. Pelleted sperm cells were resuspended using 100 µl complete buffer with detergent [15 mM Tris-HCl (pH 7.5), 60 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 0.3 M sucrose, 0.5 mM DTT, 0.5% NP-40 and 1% sodium deoxycholate] and lysed on ice for 10 min. The same volume of MNase buffer [(85 mM Tris-HCl (pH 7.5), 3 mM MgCl2, 2 mM CaCl2, 0.3 M sucrose] containing 10 units MNase (Sigma-Aldrich) was added and incubated at 37ºC for 5min. MNase treatment was stopped by adding 2 µl of 0.5M EDTA (Thermo Fisher). For STAR-ChIP, 200 µl cold 2×RIPA buffer (1% Triton, 280 mM NaCl, 0.1% SDS, 0.2% DOC, 5mM EGTA, and 1×proteinase inhibitor) was supplemented. After spinning at max speed at 4ºC for 10 min, the supernatant was transferred to a new tube and supplemented with 176 µl of RIPA buffer [10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1% Triton, 0.1% SDS, 0.1% DOC, 1 mM EDTA, 1×proteinase inhibitor]. The samples were incubated with a rabbit anti-H2Aub antibody (1:200; Cell Signaling, #8240) overnight with rotation at 4ºC. The following day, the samples were incubated with 100 µg protein A dynabeads (Life Technologies) for 2 hrs with rotation at 4 ºC. The beads were than washed five times with 150 µl RIPA buffer and once with LiCl buffer [250 mM LiCl, 10mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40, 0.5% DOC].  To elute DNA, the beads were resuspended with 200 µl TE buffer with 2.5 µl 10% SDS (Sigma-Aldrich) and 2.5 µl mg/ml Protease K (Thermo Fisher) and incubated in a shaker at 55ºC for 90 min. Supernatant was collected to a new tube and DNA were extracted by phenol-chloroform followed by ethanol precipitation. Sequencing libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs) according to the manufacturer's instructions.