title: GSM6604412: ATDC5_Day10_RUNX2_2; Mus musculus; ChIP-Seq
library_construction_protocol: Day 0, 3 and 10 ATDC5 cells were fixed with 1% formaldehyde (Electron Microscopy Sciences, Cat #: 15714) in 37 oC DEM/F12 media without FBS for 10min. Media/formaldehyde was removed and formaldehyde quenched with 155mM glycine in PBS for 10min at RT. Cells were washed 3 X 5 min 4 oC PBS. Cells were scrapped and collected in 1.5mL centrifuge tubes (300 X g, 4oC 5min) and resuspended in 750 uL ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris HCl pH8.0, protease inhibitors (Sigma-Aldrich, Cat #: P5726), Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, Cat #: P5726)). Cells were sonicated (Branson Sonifier) for 2.5 min with a 30% duty cycle and 7% power output setting. 50uL of the suspension was taken and evaluated for chromatin shearing and the concentration of chromatin/DNA per uL evaluated (Qubit 4, Life Technologies). For each ChIP assay, a volume of sample containing 50ug of chromatin was mixed with ChIP dilution buffer (0.01% SDS, 1.1% Triton X 100, 1.2mM EDTA, 16.7 mM Tris HCl pH8.0, 167mM NaCl, protease inhibitors (Sigma-Aldrich, Cat #: P5726), Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, Cat #: P5726)) to a final volume of 500uL.  A total of 5 µg of each antibody was used for each assay (mouse anti RUNX2, Santa Cruz Biotechnology, cat#: sc-390351; rabbit anti-SOX9, EMD Millipore, cat#: 05-636; rabbit anti-H3K4me3, Abcam, cat#:ab8580). A total of 2.5 µg donkey anti-mouse antibody (Jackson Scientific, UK, Cat #: 715-035-150) was used as non-specific antibody control. The mixture was incubated at 4 °C overnight with rotation, followed by binding to 50 µL Dynabead Protein A (Invitrogen, Cat #: 10006D) at 4 °C for 1 h. Samples were then treated with ChIP washing buffer I (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris pH 8.0 and 150 mM NaCl), ChIP washing buffer II (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris pH 8.0 and 500 mM NaCl) and ChIP washing buffer III (1 mM EDTA and 10 mM Tris-HCl pH 8.0). Samples were eluted with 500 µL of freshly made elution buffer (1% SDS and 0.1 M NaHCO3) for 1 h at RT. Crosslinks were reversed by adding 200 mM NaCl, 12.5 mM EDTA and 2 µL proteinase K (Invitrogen, Cat #: 25530049), followed by incubating at 65 °C for 5 h agitating at 900 rpm. DNA from each sample was extracted by phenol-chloroform and resuspended in nuclease free water. Sequencing was performed using the Illumina Novoseq protocol and generated 75bp single end reads.