GSM3759928: ChIPH3.3siG-A19.10; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% Formaldehyde in DMEM for 10 min at room temperature and quenched with Glycine at a final concentration of 125 mM. Cells were washed with PBS, pelleted by centrifugation and dry pellets were snap frozen. For nuclear extract preparation, pelleted cells were thawed on ice, resuspended in cold Swelling buffer (25 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1x protease (Roche) and phosphatase inhibitors (Sigma-Aldrich)) for 10 min and passed through a douncer 50 times. Nuclei were pelleted by centrifugation at 3000 g for 5 min at 4ºC, and resuspended in 300 ul of 1% SDS in ChIP buffer (10 mM Tris-HCl pH 7.5,150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.5 mM DTT, 1x protease (Roche) and phosphatase inhibitors (Sigma-Aldrich)). The extracts were incubated for 15 min on ice and sonicated in a Bioruptor Pico sonication device (Diagenode) for 30 cycles 30'' on/30'' off. Chromatin was cleared by centrifugation at top speed 15 min at 4ºC. For H3.3 (Millipore 09-838) ChIP, 5 ug of total chromatin was diluted 1:10 in ChIP buffer and incubated with 1 ug of antibody on rotation at 4ºC overnight. 50 ul of prewashed Dynabeads Protein G (Thermo Fisher Scientific) were added on rotation for 2h at 4ºC. Beads were washed with Low Salt buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, 1x protease (Roche) and phosphatase inhibitors (Sigma-Aldrich)), High Salt buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1% Triton X-100, 1x protease (Roche) and phosphatase inhibitors (Sigma-Aldrich)) and eluted by incubating in a thermomixer with Elution buffer (1% SDS, 100 mM NaHCO3) for 30 min at 65ºC and 1000rpm. Samples were reverse-crosslinked by incubating at 65ºC overnight and incubated with Proteinase K for 1h at 45ºC. Chipped DNA was purified using the MinElute PCR Purification Kit (Qiagen) and eluted in 40 ul. Library construction was done with the kit NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, catalog # E7645), following manufacturer´s instructions and 9-10 cycles of amplification
GEO Accession
GSM3759928