GSM3336807: Input for dsCP190 treatment, replicate 2; Drosophila melanogaster; ChIP-Seq
2-3 X 10^7 cells were fixed by adding 1% formaldehyde directly to cells in culture medium for 10 min at RT with gentle agitation. Then formaldehyde was quenched with 0.125 M glycine with gentle agitation for 5 min at RT. Cells were pelleted by centrifugation at 2000 x g and washed twice with ice-cold PBS. Pellets were resuspended in 0.8 mL ice-cold cell lysis buffer [5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40, supplemented with Complete protease inhibitors (Roche)], incubated on ice for 10 min, then pelleted by centrifugation at 2000 x g for 5 min at 4°C. Next, the supernatant was removed and pellet was resuspended in 1 mL nuclear lysis buffer [50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, supplemented with Complete protease inhibitors (Roche)] and incubated for 10 min at 4°C on a rotator. Afterwards, 0.5 mL of IP dilution buffer [16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS, supplemented with Complete protease inhibitors (Roche)] and 300 mg of acid washed 212-300 micron glass beads (Sigma) were added to the lysate, and chromatin was fragmented to an average size range of 200-500 bp using a Bioruptor (Diagenode) using 10 cycles of 30 s on and 30 s off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at -80°C. Chromatin was diluted to 1:5 with IP dilution buffer and added to 50 µL prewashed Protein A-sepharose beads (GE) and 5 µg of respective antibody and rotated overnight at 4°C. The next day, beads were washed with the following wash buffers: three times with Low salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), three times with High salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), and two times with LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% Deoxycholate). Chromatin was eluted twice with 200 μL of elution buffer (0.1M NaHCO3, 1% SDS) for 30 min at 65°C each in a thermomixer at 800 rpm. Decrosslinking solution (20 μL of 5M NaCl, 8 μL of 0.5M EDTA, 10 μL of 1M Tris-HCl pH 8) was added to the eluates and further incubated overnight at 65°C. After de-crosslinking, samples were treated with 4 µL of Proteinase K (20 mg/mL) for 2 h at 50°C and then purified by Phenol-Chloroform followed by ethanol precipitation with 0.1 vol of 3M NaOAc pH 5.2 and 2.5 vol of 100% ethanol supplemented with 2 μL of Glycoblue (Ambion). After incubating overnight at -80°C, samples were centrifuged 20 min at 10,000 x g at 4°C. Pellets were washed with 70% ethanol. Pellets were air dried at RT prior to resuspension in 10 μL of nuclease free water. Libraries were constructed by pooling two IP samples using TruSeq adapters (Illumina) according to the TruSeq Illumina ChIP-seq sample preparation protocol with the following modifications: after adaptor ligation and PCR amplification samples were purified by using AMPure XP Beads (sample to beads ratio 1:0.8) according to manufacturer's protocol.
GEO Accession
GSM3336807