GSM1200247: S2_DsxF; Drosophila melanogaster; ChIP-Seq
Formaldehyde was added to a final concentration of 1 % to cross-link proteins to DNA. The fixation reaction was incubated on a shaker for 10 min at room temperature. The reaction was quenched by adding glycine to a final concentration of 125 mM and incubation for 5 min on a shaker at room temperature. Subsequently the cells were washed twice with ice-cold PBS. After centrifugation at 500 x g (1680 rpm) for 5 min at 4 °C the cell pellet was resuspended in 10 ml ice-cold cell lysis buffer (5 mM pH8.0 PIPES buffer, 85 mM Potassium chloride, 0.5% Nonidet P40 and protease inhibitors (cOmplete, EDTA-free, Roche, indianapolis, IN)) for 10 minutes at 4 °C. Nuclei were released by douncing with a Wheaton homogenizer (pestle B). The crude nuclear extract was collected by centrifugation at 500 x g (1680 rpm) for 5 min at 4 °C, resuspended in 2 ml ice–cold nuclear lysis buffer (50 mM pH8.1 Tris.HCl, 10 mM EDTA, 1 % SDS and protease inhibitors) and incubated for 20 minutes at 4 °C. After adding 1 ml ice-cold IP dilution buffer (0.01 % SDS, 1.1 % TritonX-100, 1.2 mM pH 8 EDTA.Na2, 16.7 mM pH8 Tris.HCl, 167 mM NaCl and protease inhibitors) and 0.3 g acid-washed glass beads (Sigma-Aldrich, St. Louis, MO) to the nuclear extract, the chromatin was sheared to 200-1000 bp using a Misonix Sonicator 3000 (Misonix, Inc. Farmingdale, NY). Sonication was performed on ice water with 8 pulses of 30 seconds and 30 second intervals. Thereafter the cell debris was removed by centrifugation at 16000 x g (13000 rpm) for 10 min at 4 °C. Aliquots of sonicated chromatin were stored at -80 °C. To assess chromatin quality, aliquots of the chromatin (100 μl) were treated with 40 µg proteinase K (Invitrogen, Carlsbad, CA) for 2 h at 50 °C after the DNA-protein complexes were de-crosslinked at 65°C overnight. After purification of the DNA by phenol-chloroform extraction and ethanol precipitation 3 μl aliquots were subjected to electrophoresis and the chromatin fractions with optimal size (200-1000 bp) range were subjected to immunoprecipitation experiments. Libraries were made using the Genomic DNA sample preparation kit (Illumina). Adapter-ligated DNA of 200 ± 25 bp range was excised from the gel before PCR amplification.