GSM4983391: ChIP HP1 IP repl2 in ovaries; Drosophila melanogaster; ChIP-Seq
Ovaries were snap frozen in batches of and stored at -80 ˚C until further processing.  Each batch was transferred to ice and homogenized for 1 min with an electric grinder in 50 µl of buffer A (60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES [pH 7.6], 0.5 % TritonX100, 0.5 mM DTT, 10 mM Sodium Butyrate, 1X  Protease Inhibitors) supplemented with 1.8% PFA. The homogenate was filled up to 1 ml with buffer A + 1.8% PFA crosslinked for another 14 min rotating at room temperature. The crosslinking was stopped by the addition of 2.5 M glycine (250 mM final concentration). The nuclei were isolated by centrifugation and washed twice with buffer A. Subsequently the nuclei were resuspended in 100 µl Lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 1% SDS, 0.5% N-Laurosylsarcosine,1X  Protease Inhibitors) and incubated for 2 hrs at 4 ˚C on a rocking plate. After that, the chromatin was sheared in a picoraptor (Diagenode) for a total of 16 cycles (30 sec on/30 sec off). Debris was pelleted at 20000 xg for 5 min at 4 ˚C and the supernatant was transferred to a new tube and diluted with 900 µl  Lysis buffer (w/o SDS) to a total SDS concentration of 0.1%.    Chromatin was precleared by addition of 50 μl of Protein A-Agarose (PA) suspension (Roche 11134515001) followed by overnight incubation at 4°C. PA was removed by centrifugation, antibodies (5 µl of rabbit anti-HA (C29F4) Cell Signaling) were added to the supernatant, and samples were incubated for 4 hr at 4°C in a rotating wheel. PA (50 μl) was added, and incubation was continued overnight at 4°C. Antibody-protein complexes were collected by centrifugation at 4,000 rpm for 1 min, and the supernatants were discarded. Samples were washed four times in Lysis buffer (with only 0.05% SDS) and twice in 1 mM EDTA, 10 mM Tris (pH 8) buffer (each wash, 5 min at 4°C). Chromatin was eluted from PA in 150 μl of 10 mM EDTA, 1% SDS, 50 mM Tris (pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. The eluate was incubated overnight at 65°C to reverse crosslinks and treated with Proteinase K for 3 hr at 50°C.  The chromatin was purified using ChIP DNA Clean & Concentrator columns (Zymo Research) and libraries were generated using the NEB Ultra II library preparation kit (E7645S). libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions