GSM7157607: Differentiated Spermatogonia H3K36me3 Dnmt3a f/- ; Dnmt3b f/- ; Stra8-iCre tg rep 2; Mus musculus; ChIP-Seq
For spermatogonia, testicular cell suspension was prepared by incubation of seminiferous tubules in 200 U/ml Collagenase type I (Worthington Biochemical LS004196), 5 μg/ml DNAse I (Roche 10104159001), and 0.05%Trypsin (Gibco 25200056) in GBSS (Sigma G9779) as described in (60). Cells were stained with 1/200 anti-CD117/c-kit PE (eBioscience 12-1171-83), anti-CD324/E-Cadherin eFluor 660 (eBioscience 50-3249-82) and anti-CD49f/Integrin alpha 6 PE-Cyanine7 (eBioscience 25-0459-82) for 1 hour at 32oC with constant shaking and protected from light. After washing cells were incubated with 20 μg/ml with Hoechst 33342 (Thermo Fischer Scientific H3570) for 1 hour at 32oC with constant shaking and protected from light and then with 30nM DRAQ7 (Biostatus DR71000) for 5 minutes on bench. The stained cells were strained through a 40 μm nylon filter into 5 ml polypropylene tubes and were sorted on a BD FACSAria III cell sorter fitted with a 70 μm nozzle (Becton Dickinson) using a 375nm laser to excite Hoechst 33342, a 561nm laser to excite PE and PE-Cy7 and a 633nm laser to excite DRAQ7 and eFluor600. Cells were first gated for FSC and SSC to exclude debris. Then the live cells were selected based on the absence of DRAQ7 signal detected using 755LP, 780/60BP. Then we gated for cells positive for Hoechst 33342 emission which was detected using a 670LP (Hoechst-Red) and 450/20 BP (Hoechst-Blue). We gated for the Hoechst-Redlow and Hoechst-Bluemid population which is enriched for 2N spermatogonia. Fluorescence for PE was detected using a 582/15BP filter, for PE-Cy7 was detected using 735LP,780/60BP filter and for eFluor660 using a 660/20BP filter. Undifferentiated spermatogonia were sorted as CD324high, CD49fhigh, CD117low and differentiated spermatogonia were sorted as CD324low, CD49flow, CD117high. Briefly 5000-7500 spermatogonia cells were FACS-sorted directly in 20 μl nuclear isolation buffer (Sigma-Aldrich NUC101) in Eppendorf® LoBind microcentrifuge tubes. The nuclei were centrifuged at 500g for 5 minutes at 4°C in a swing bucket centrifuge and the volume of the sample was reduced to 10 μl. The sample was snap frozen in liquid nitrogen and stored at –80°C freezer until further processing. The sample was thawed on ice and 1 μl of TS buffer (5% Triton X-100, 5% Sodium deoxycholate) was added for 10 minutes to ensure permeabilization of nuclei. 40 μl of MNAse digestion buffer (3.33 gelUnits/μl Micrococcal Nuclease (NEB M0247S), 1.25X Micrococcal Nuclease buffer, 6.25% PEG 6000, 0.85mM DTT) was added to the sample for 10 minutes at 25 °C to fragment the chromatin. Reaction was stopped by addition of 5 μl 100 uM EDTA and the sample was placed on ice. The sample was diluted by addition of 145 μl ChIP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1X EDTA-free protease inhibitor cocktail). To ensure release of chromatin fragments, the sample was sonicated using the Diagenode Bioruptor Plus® for 3 cycles of 5 seconds ON and 30 seconds OFF at low output. Then 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 0.75 μg anti-H3K36me3 (Cell Signaling #4909) 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 2X volumes of Ampure XP DNA purification beads (Beckman Coulter, A63881) and resuspended in 25 μl nuclease free H2O. NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645L)