Third instar larvae were dissected in cold PBS and imaginal disc complexes (anterior one third of the larvae after removing the fat body and salivary glands) were fixed in 1 ml fixation buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5; 1 mM ethylenediaminetetraacetic acid [EDTA]; 0.5 mM ethylene glycol tetraacetic acid [EGTA]; 100 mM NaCl; 2% formaldehyde) for 30 min at room temperature. Fixed disc complexes were washed 3x fast and 2x 20 minutes with PBST (PBS, pH 7.4; 0.1% Triton X-100; 0.1% Tween-20), and were stored at 4 C until enough discs were obtained. 100-500 wing discs were dissected away from the cuticle and resuspended in buffer A2 (15 mM HEPES, pH 7.5; 140 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1 % sodium dodecyl sulfate [SDS]; 0.5 % N-lauroylsarcosine; 1 Roche complete protease inhibitor cocktail, cat. no. 5056489001). Tubes were flash frozen in liquid nitrogen and stored at -80 C. Imaginal discs were pooled to reach 500 wt discs (or 100 mutant), and sonication was performed in a Bioruptor sonicator for 5 min (30 s on/off cycle at the high setting) in buffer A2. Following centrifugation (16,000 g; 10 min at 4 C), the supernatant containing soluble chromatin was transferred to fresh tubes, and used for ChIP-nexus. Library preparation was done as described in He, Johnston and Zeitlinger, 2015 Nature Biotechnology.