GSM7157586: Sperm H3.3 Dnmt3a f/- ; Dnmt3b f/- ; Stra8-iCre tg rep 1; Mus musculus; ChIP-Seq
For sperm cell sorting, mouse cauda epididymides were dissected into a Petri dish and fat patches were removed with forceps and scissors. Each epididymis was punctured with a needle and carefully squeezed into 100 ul of PBS with the help of two forceps. Sperm was transferred into an Eppendorf tube and was allowed to liquefy at room temperature for 5 minutes. To break sperm tails, cells were briefly sonicated with a Brandson Tip digital sonicator with 10% amplitude and 3 cycles of 0.5 sec ON / 2 sec OFF. Cells were stained with 2 ul/ml Hoechst 33342 (H3570, ThermoFisher) for 1h at 25°C with constant shaking and protected from light. Sperm cells were filtered through a 40 μm Nylon filter into 5 ml polypropylene tubes.Sperm cells (500.000 per reaction) were FACS sorted in Eppendorf® LoBind microcentrifuge tubes. The cells were pelleted at 6000g for 5 minutes at 4°C in a benchtop centrifuge. The pellet was snap frozen in liquid nitrogen and stored at –80°C freezer until further processing. The pellet was thawed on ice and resuspended in 500 μl PBS. 50mM DTT was added for 2 hours at 21°C to reduce disulfide bonds of protamine. 100mM N-Ethylmaleinimid was added for 30 minutes at 21°C to modify the cysteine residues. The cells were pelleted at 6000g for 5 minutes at 4°C in a benchtop centrifuge and washed once with 600 μl PBS. Cells were resuspended in 50 μl Lysis buffer (20 mM Tris-HCl pH 7.5, 60 mM KCl, 5mM MgCl2, 0.1 mM EGTA, 300 mM Sucrose, ,0.5 mM DTT, 0.25 % Igepal, 0.5% sodium deoxycholate, 1X EDTA-free protease inhibitor cocktail) and incubated for 30 minutes at 21°C. 50 μl of MNAse digestion buffer (5 gelUnits/μl Micrococcal Nuclease (NEB M0247S), 2X Micrococcal Nuclease buffer) was added to the sample for 15 minutes at 37°C to fragment the chromatin. Reaction was stopped by addition of 5 μl 500 uM EDTA and the sample was centrifuged at 16000g for 10 minutes at 4°C in a benchtop centrifuge. The supernatant chromatin was pre-cleared with 10 μl of 1:1 protein A: protein G Dynabeads (Thermo Fisher Scientific, 10001D and 10003D). The precleared chromatin sample was incubated with 0.5 μg of anti-H3K4me3 (Millipore 17-614) or 1 μg anti-nucleosome (44) provided by J. van der Vlag) or 1 μg anti-H3.3 (Cosmo Bio CE-040B) or 1 μg anti-H3.1/2/t (63) provided by J. van der Vlag) conjugated with 10 μl of 1:1 protein A:protein G Dynabeads overnight at 4 °C on a rotator. Chromatin-Antibody-Dynabeads complexes were washed once with 500 μl of ChIP buffer, 3 times with 1ml of Low Salt wash buffer (20 mM Tris-HCl (pH 8.0), 0.05% SDS, 1% Triton X-100, 2 mM EDTA and 150 mM NaCl) and 3 times with 1 ml High Salt buffer (20 mM Tris-HCl (pH 8.0), 0.05% SDS, 1% Triton X-100, 2 mM EDTA and 250 mM NaCl). Chromatin was eluted in 40 μl of Elution buffer (100 mM NaHCO3 and 1% SDS) for 90 minutes at 65 °C. DNA from eluted material was purified by phenol-chloroform, ethanol-precipitation and resuspended in 25 μl nuclease free H2O. NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645L)