40% amplitude for 0.7 s on and 1.3 s off for 6 minutes with the thermal block set at −6 ˚C to generate the optimal majority fragment size range between 200 and 600 bp. Samples were then centrifuged to pellet debris and 500ul of the supernatant – which is roughly half of the total volume-was incubated overnight at 4 ˚C with 2.5uL of the antibody with a final volume of 3 mL using the ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.1, 167 mM NaCl). Chromatin labeled with the H3K9Ac mark and bound to the antibody was purified with protein A sepharose beads, and the captured chromatin fragments were reverse cross-linked overnight in 250 mM Tris-HCl, pH6.5, 62.5 mM EDTA pH8.0, 1,25M NaCl, 5 mg/mL Proteinase K, 62.5 ug RNaseA at 65˚C. The captured DNA fragments were then extracted using a phenol:chloroform phase separation, and prepared for library construction using the END-IT DNA repair kit (Epicenter Cat. No. ER0720), and single 3′-adenine overhangs were added using Klenow(3′-5′ exo-) (New England Biolabs, Cat. No. M0212L). Qiagen MiniElute spin columns were used to clean up each of these reactions. Barcoded Illumina adapters prepared by the
