Isolation and Purification of Drosophila Nuclei

Adult flies were anesthetized by CO₂ and flash frozen in liquid N₂. Heads were separated from thoracicoabdominal segments, wings and legs by vigorous vortexing followed by separation over dry ice cooled sieves. In all, 600–10 000 frozen heads were added to 100 ml of 10 mM β-glycerophosphate pH 7, 2 mM MgCl₂, 5 mM sodium butyrate, 1X complete protease inhibitor cocktail (Roche: 11873580001), and the suspension was passed over a Yamato continuous flow homogenizer, set at 100 rpm, five to seven times. The homogenate was filtered over Miracloth (EMD Biosciences: 475855) and brought to 0.7 mM β-mercaptoethanol and 0.5% NP-40. After six tractions in two 40 ml Dounce homogenizers (tight-pestle B), 600 μl of antibody-adsorbed beads were added to 100 ml of lysate. The binding reaction was performed at 4°C for 30 min with constant end-over-end agitation. Beads were then collected on a magnet (Invitrogen: 123-02D) and washed three to four times in 50 ml 10 mM β-glycerophosphate pH 7, 250 mM sucrose, 2 mM MgCl₂, 25 mM KCl and 5 mM sodium butyrate. Bead-bound nuclei in 20 ml of wash buffer were then passed over a 20 um nylon mesh (Small Parts: B001D8ECDE), returned to the magnet stand and resuspended in 1 ml of 10 mM β-glycerophosphate pH 7, 250 mM sucrose, 2 mM MgCl₂, 25 mM KCl and 5 mM sodium butyrate. Sodium butyrate and the protease inhibitor cocktail are omitted from all buffers, if nuclei were to be used for transcript profiling (RNA-seq).