GSM6751579
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with glycine at a final concentration of 0.125 M. Chromatin was sheared using ultrasonication (Covaris S2). A total of 2.5 µg of antibody against H3K27ac (Abcam, ab4729), or FLAG (M2, Sigma Aldrich, F3165), was bound to 20 µl pre-washed Dynabeads Protein A (Thermo Fisher Scientific) in a total volume of 200 µl PBS containing 0.02% Tween-20 for 1h at room temperature rotating at 6 rpm. The buffer was removed on a magnet and antibody-coupled beads were resuspended in 120µl dilution buffer (20mM Tris pH7.4, 2mM EDTA, 100mM NaCl, 0.5% Triton x-100) containing proteinase inhibitors (cOmplete™, EDTA-free Protease Inhibitor Cocktail, 50x, Sigma). 80µl sonicated chromatin was added to the antibody-coupled Dynabeads (sonicated chromatin of approx. 1.7x106 cells) and incubated for 3h at room temperature rotating at 6 rpm. Beads were washed on a magnet and chromatin was eluted. After crosslink reversal, RNase A and proteinase K treatment, DNA was extracted with the Monarch PCR & DNA Cleanup kit (NEB). Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to manufacturer's instructions