GSM3263394: input of CDK9 IP, siAGO2 treatment, replicate 2; Drosophila melanogaster; ChIP-Seq
Approximately 1-3 X 10^7 cells were fixed by addition of 1% formaldehyde to cell media for 10 min at RT with gentle agitation. Formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. Cells were pelleted at 2000 xg, washed twice in PBS, and 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 10 min, and pelleted by centrifugation at 2000 xg for 5 min at 4°C. Next, the supernatant was removed and pellets were resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA.Na2, 1% SDS) and incubated for 10 min at 4°C. Afterwards, 0.5 mL of IP dilution buffer was added (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS), and chromatin was fragmented to an average size of 300 bp by using PicoBioruptor (Diagenode) using 10 cycles of 30 s on plus 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 buffer, and the assayed antibody in addition to 50 μL of prewashed protein A/G 50% slurry was added and rotated overnight at 4°C. The next day, beads were washed first three times with low salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS); next three times with high salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS); finally two times with LiCl buffer (10 mM Tris-HCl pH 8, 1 mM EDTA.Na2, 250 mM LiCl, 1% NP-40, 1% DOC). Chromatin was eluted twice with 200 μL of elution buffer (500 μL of 1M NaHCO3, 250 μL of 20% SDS, 4.25 mL dH2O) for 30 min at 65°C each and further incubated overnight at 65°C with 38 μL of decrosslinking solution (20 μL of 5M NaCl, 8 μL of 0.5M EDTA, 10 μL of 1M Tris-HCl pH 8). After de-crosslinking, samples were treated with Proteinase K for 2 h at 50°C and then combined with 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 5 min at 10,000 xg . The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and subsequently precipitated 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 30 min at -80°C, samples were centrifuged 20 min at 4°C at 10,000 xg. Pellets were washed with 70% ethanol and centrifuged 5 min at 4°C at 10,000 xg. Pellets were air dried at RT prior to resuspension in 10 μL of dH2O. Samples for ChIP-seq were prepared according to the manufacturer's protocol with TruSeq adapters (Illumina).