GSM6934452: AW_Control, replicate 1; Drosophila melanogaster; ChIP-Seq
75 wandering third-instar larvae (3 biological replicates per genotype) were collected in a 2ml DNA LoBind Eppendorf tube and washed twice with 1ml 1X PBS. The larvae were homogenized in ice-cold lysis buffer (200ul 1X protease inhibitor cocktail, 250ul PMSF, 800ul 1X PBS, 1ul Tween 20) using a pellet pestle. The homogenized lysate was supplemented with 244.5ul of 11% formaldehyde to a final concentration of 1.8% and the samples were crosslinked for 15 minutes at room temperature on a rotator. Glycine was added to a final concentration of 500mM to quench the fixative on ice for 5 minutes at room temperature. The larval debris was pelleted at 1,000g for 3 minutes and the supernatant was removed. The pellet was resuspended in 1ml sonication buffer (0.5% SDS, 20mM Tris pH 8.0, 2mM EDTA, 0.5mM EGTA, 0.5mM PMSF, 1X protease inhibitor cocktail) and chromatin was fragmented to 300 – 500bp Bioruptor sonicator (UCD-200) for 20 cycles (30 seconds high frequency sonication, 1.5 sec pause) in a cold room. The sonicated material was pelleted at 10,000g for 10 minutes at 4°C, supernatant was collected, then fragment size was checked prior to immunoprecipitation. The sonicated chromatin was pre-cleared and incubated with anti-GFP antibody overnight (TP-401, Torrey Pines Biolabs) 4°C overnight. The immunoprecipitated chromatin was then collected with pre-washed Protein A agarose beads for 2 hours. The beads were sequentially washed with the following buffers: 1 low salt buffer wash (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCL pH 8.0, 150mM NaCl), 3 high salt buffer washes (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCL pH 8.0, 500mM NaCl), 1 LiCL wash (2mM EDTA, 20mM Tris-HCl pH 8.0, 0.25M LiCl, 1% NP-40) and 2 TE buffer washers before elution. Bound chromatin on beads was eluted twice at room temperature using 250ul of freshly prepared ChIP elution buffer (1% SDS, 100mM NaHCO3) for 15 minutes and reverse-crosslinked overnight. The eluates were then treated with RNase A and proteinase K prior to DNA extraction via phenol-chloroform extraction and ethanol precipitation. Libraries were made and sequenced at Novogene. Libraries were made using NEBNext Ultra II DNA Library Prep Kit at Novogene