GSM9053457: H3K27M_wing_H3K27me3_rep_1; Drosophila melanogaster; OTHER
Imaginal wing and eye-antennal discs were harvested from crawling third-instar larvae. Larvae were staged to within 116 and 119 hours after egg laying. For each replicate, twenty discs were dissected into ice cold 1X PBS solution. Two biological replicates were dissected for each genotype.. Samples were washed three times in Wash Buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5 mM spermidine (Sigma S0266-1G), and Pierce EDTA-free Protease Inhibitor (ThermoFisher PIA32955)) and incubated with activated concanavalin-A (ConA) paramagnetic beads (EpiCypher SKU: 21-1401) for ten minutes in PCR strip tubes. Samples were resuspended in cold Antibody Buffer (Wash Buffer + 0.05% digitonin (Sigma Millipore 300410250MG) and 2mM EDTA (Fisher S311500)). 2 uL of SNAP-CUTANA K-MetStat nucleosomes (EpiCypher SKU: 19-1002) diluted 1:100 in Wash Buffer were added to each sample for spike-in normalization. Antibody was added to samples at a 1:50 concentration and incubated overnight at 4ºC (Rabbit anti-H3K27me3 (Cell Signaling Technology #9733S) or rabbit IgG control (ThermoFisher #10500C)). The following day, discs were washed with Digitonin Buffer (Wash Buffer + 0.05% digitonin (Sigma Millipore 300410250MG)), then incubated with pAG-MNase (EpiCypher SKU: 15-1016) for ten minutes to allow protein A/G binding to antibody. 1 uL of cold 100 mM CaCl2 was added to samples to activate pAG-MNase and incubated on nutator for 2 hours at 4ºC. Reaction was quenched with Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A , 50 µg/mL Glycogen). Chromatin was eluted from samples at 37ºC for ten minutes, then DNA was purified with the Qiagen MinElute Reaction Cleanup Kit (Qiagen #28204). Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina