GSM7287699: 2-3H_PQ-mut_input_rep1; Drosophila melanogaster; ChIP-Seq
ChIP was performed as described previously (Blythe and Wieschaus 2015) on hand selected 2-3 hr AEL embryos from: sfGFP-GAF/+ and sfGFP-GAFSDPQ/sfGFP-GAFSDPQ females. Briefly, 200-400 embryos were collected, dechorionated in 50% bleach for 3 min, fixed for 15 min in 4% formaldehyde and then lysed in 1 mL of RIPA buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 150 mM NaCl). The fixed chromatin was then sonicated for 20 s 11 times at 20% output and full duty cycle (Branson Sonifier 250). Chromatin was incubated with 6 μg of anti-GFP antibody (Abcam #ab290) overnight at 4°C , and then bound to 50 μl of Protein A magnetic beads (Dynabeads Protein A, Thermo Scientific). The purified chromatin was then washed, eluted, and treated with 90 μg of RNaseA (37°C, for 30 min) and 100 μg of Proteinase K (65°C, overnight). The DNA was purified using phenol/chloroform extraction and concentrated by ethanol precipitation. Each sample was resuspended in 25 μl of water. Sequencing libraries were made using the NEB Next Ultra II library kit. Libraries were sequenced on the Illumina NextSeq 500 using 75bp single-end reads at the Northwestern Sequencing Core (NUCore).