For each sample, cells were trypsinized for 8–10 min, trypsin was quenched with 10 mL of ES media, and 107 cells were obtained. Cells were spun down, washed with 10 mL PBS, fixed for 12 min in a mix of formaldehyde (to a final concentration of 1%) and Fix Buffer (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl), and then quenched by glycine (final concentration of 0.125 M). Cells were incubated on ice, and then spun down at 1000g for 5 min. Nuclei were prepared by consecutive washes with Rinse 1 Buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), Rinse 2 Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl), and Shearing Buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris HCl pH 8). After a final spin down, cells were resuspended in 100 μL of Shearing Buffer and 1× protease inhibitor cocktail (Calbiochem) nanodroplets (generous gift from Samantha Pattenden),40 (link) and sonicated in an E110 Covaris Sonicator for 4 min or until DNA was sheared to between 70bp and 500bp (as confirmed by agarose gel).
After sonication, the rest of the protocol was performed with a ChIP-IT High Sensitivity Kit (Active Motif, 53040), and an H3K27ac antibody was used for the pull down (Abcam, ab4729).
After sonication, the rest of the protocol was performed with a ChIP-IT High Sensitivity Kit (Active Motif, 53040), and an H3K27ac antibody was used for the pull down (Abcam, ab4729).
