For each sample and replicate, 50 million 80-90% confluent S2 cells were harvested, washed with PBS, cross-linked in 1% formaldehyde at room temperature for 10 min and quenched with 125mM glycine. For nuclei isolation, cells were incubated in cell lysis buffer (50mM Tris pH8, 2mM EDTA, 0.5% NP-40, 10% glycerol, cOmplete Protease Inhibitor Cocktail (Roche)) for 10min at 4°C. After pelleting, nuclei were lysed in 50mM Tris pH8, 10mM EDTA, 1% SDS and cOmplete Protease Inhibitor Cocktail (Roche) for 15min. Chromatin fragments between 200-1000bp were obtained through sonication (E220 Focused-ultrasonicator, Covaris). Chromatin from approximately 30 million cells was immunoprecipitated overnight at 4°C with the anti-FLAG M2 (Merck, F1804) antibody. Antibody-bound chromatin was loaded on 40ul magnetic Dynabeads (1:1 mix of Protein G and A beads, Invitrogen) for 1h 4°C, washed twice in low-salt, high-salt, LiCl and TE buffer and eluted with 1% SDS, 100mM NaHCO3, 5mM DTT in water. Subsequently, samples were decrosslinked overnight at 65°C and treated with RNase A and proteinase K. DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo Research), according to manufacturer's instructions. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer's protocol.