title: GSM8800243: K27m3_SgU_WT_r2; Mus musculus; ChIP-Seq
library_construction_protocol: To profile histone modifications in spermatogonia cells we followed the previously published ULI-NChIP protocol (Brind'Amour, Liu et al. 2015) with minor modifications. 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 or 1 μg anti-H3K36me2 or 1 μg anti-H3K27me3 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. For library construction the ChIP purified DNA was used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645L) with modifications of the manufacturer instruction as we assembled half volume reactions and we doubled reaction incubation times. For adaptor ligation we used 1:15 adaptor dilution. We amplified the ligated fragments for 12 cycles using single indexed primers.