Chromatin Immunoprecipitation of Drosophila Larvae

Third instar larva WID or EID isolated from Canton S flies were fixed, pooled in 700 μL and processed as described^{17 (link)}. Around 300 imaginal discs were used in these experiments. Trypsin treated cells from GFP transgenic flies were fixed after sorting for 10 minutes at room temperature and sonicated in a Diagenode Bioruptor for 15 minutes at high power in lysis buffer (1% SDS, 10 mM Tris HCl ph 8.0 and 2mM EDTA). Immunoprecipitations were performed in RIPA buffer. For L3 ChIPs and Imaginal Discs ChIPSeq experiments we used 1 μg of the corresponding antibody. For ChIPs in sorted cells we used 0.45 μg of anti-H3K4me3, 0.3 μg of anti-H3K36me3, 0.33 μg of anti-H3K27ac and 1 μg of anti-H3K27me3. For L3 time-specific ChIPs, 5 Canton S wall-wandering third instar larvae were disrupted, fixed and sonicated as indicated above. . Immunocomplexes were recovered with Invitrogen ProteinA magnetic beads for 2h. The beads were washed three times in RIPA or IP buffer, once in LiCl buffer and twice in TE^{17 (link)}. Primers used for Real-Time PCR are listed in Supplementary Table 11. The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).