y w ubxflp/ y w; FRT82B M(3) ubiGFP/ FRT82B cicQ474X wts149
Sequenced DNA Library
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.