70 to 130 million D. melanogaster Kc167 cells, resuspended in 20 ml of complete Schneider's Drosophila Medium, were crosslinked by adding 1:10 of the volume of Fixing Solution (100 mM NaCl, 50 mM Hepes pH 8, 1mM EDTA, 0.5 mM EGTA, 10% methanol-free formaldehyde) and rotated at room temperature for 8 min. 1.17 ml of freshly-prepared 2.5 M glycine was added to stop the fixation (final conc. 125 mM). Cells were pelleted at 500 g for 10 min (4°C) and resuspended in 10 mL of ice-cold PBS. 3.5 million of fixed D. virilis cells (fixed as described for D. melanogaster cells) were added for every 70 million D. melanogaster cells. Cells were pelleted at 526 g for 10 min (4°C) and resuspended in 1 ml of PBS + 0.5% Triton-X-100 + 1X cOmplete EDTA-free Protease Inhibitor for every 70 million D. melanogaster cells and rotated at 4°C for 15 min to release nuclei. Nuclei were pelleted at 2000 g for 10 min and washed once with 10 ml of ice-cold PBS. Nuclei were suspended in 1 ml of RIPA buffer (10 mM Tris-HCl pH 8, 140 mM NaCl, 1mM EDTA, 0.1% Na-deoxycholate, 1% Triton-X-100, 0.1% SDS, 1 mM PMSF, 1X cOmplete EDTA-free Protease Inhibitor) + 2 mM CaCl2 for every 70 million D. melanogaster cells, divided into 1 ml aliquots and flash-frozen in liquid N2. 1 mL of fixed nuclei was quickly thawed and 1 µL of MNase (to 0.6 units) (Sigma-Aldrich, Cat. No N5386) added. Chromatin was digested for 35 min at 37°C. MNase digestion was stopped by transferring the samples on ice and adding 22 µL of 0.5 M EGTA. Samples were mildly sonicated using a Covaris S220 instrument with the following settings: 50 W peak power, 20% duty factor, 200 cycles/burst, 8 min total time. Insoluble chromatin was removed by centrifugation at 16,000 g for 30 min at 4°C. Soluble chromatin was pre-cleared by incubation with 10 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry (GE Healthcare, Cat. No 17-5280-11 and 17-0618-06) for every 100 µL of chromatin for 1 h at 4°C. 100 µL of pre-cleared chromatin were set aside (input) and kept overnight at 4°C, while each primary antibody was added to 300 µL of chromatin and incubated overnight at 4°C. 40 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry was added for each immunoprecipitation and rotated 3 h at 4°C. Beads were washed 5 times with 1 ml of RIPA (5 min rotation at 4°C, pelleted at 3000 g for 1 min between washes) and resuspended in 100 µL of TE (10 mM Tris pH 8, 1 mM EDTA). 0.5 µL of RNaseA (Sigma-Aldrich, Cat. No. R4875) was added to both input samples and resuspended beads, followed by incubation at 37°C for 30 min. After addition of 6 µL of 10% SDS, protease digestion (250 ng/ µL Proteinase K, Genaxxon, Cat.no. M3036.0100) and crosslink reversal were performed simultaneously at 68°C for 2 hr. DNA was purified using 1.8X Agencourt AMPure XP beads (Beckman Coulter, Cat No A63880) following the standard protocol and eluted in 30 µL of 5 mM Tris-HCl pH 8. Libraries for sequencing were prepared using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, E7465). Libraries were sequenced on an Illumina HiSeq 1500 instrument at the Laboratory of Functional Genomic Analysis (LAFUGA, Gene Center Munich, LMU).