GSM4212625: E93GOF-3LW-Wing-antiHA-ChIP-Rep1; Drosophila melanogaster; ChIP-Seq
200+ Wings were manually dissected from 3LW male and female UAS-E93-3xHA larvae. Wings were fixed in batches of 20 in 4% paraformaldehyde, 50mM HEPES, 100mM NaCl, 1mM EDTA, 0.5mM EGTA for 20-minutes at room temperature, followed by quenching with 125mM glycine in 1x PBS, 0.01% Triton. Fixed wings were Dounce homogenized in 10mM HEPES, 10mM EDTA, 0.5mM EGTA, 0.25% Triton, 1mM PMSF. Nuclei were pelleted at 4,500xg for 20-minutes, and re-suspended in 10mM HEPES, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton, 1mM PMSF. After nutating at 4C for 10-minutes, nuclei were pelleted again and re-suspended in 140mM NaCl, 10mM HEPES, 1mM EDTA, 0.5mM EGTA, 1mM PMSF, 0.1% SDS, followed by sonication on ice with a Branson Sonifier until average chromatin fragment size was 200bp. The soluble chromatin fraction was used for ChIP. Briefly, extracts were pre-cleared with protein-A dynabeads for 2 hours at 4C, and cleared extracts were incubated with 5 µg of Rabbit anti-HA (Abcam, ab9110) overnight at 4C. Bead pulldown was performed for 3-hours the following day. Antibody-bead complexes were washed successively, and then eluted with 1% SDS, 250mM NaCl, 10mM Tris, 1mM EDTA. Samples were treated with RNase A and proteinase K, heated overnight at 65C to reverse crosslinks, and purified DNA was recovered by phenol-chloroform/ethanol precipitation. Rubicon Thruplex DNA-seq kit
GEO Accession
GSM4212625