GSM1419183: rox_MOF; Drosophila melanogaster; ChIP-Seq
We used 125 salivary glands for each sample. The glands were dissected in PBS and directly transferred to 2% formaldehyde in PBS with 0.1% Triton X-100 for 1–2 min for fixation. The glands were then washed in PBS and glycine was added to a final concentration of 0.125 M. The glands were then washed once in PBS, 1 mM PMSF, protease inhibitor cocktail (Roche) followed by two washes in TBS, 1 mM PMSF, and protease inhibitor cocktail. The samples were homogenized in 250 μl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1]). Approximately 60 μl glass beads were added prior to sonication for 2 × 10 sec at output level 1, 2 × 10 sec at output level 2, 2 × 10 sec at output level 3 and finally 2 × 10 s at output level 4 (Misonix XL2020, microtip). The samples were cleared by centrifuging for 10 min at 16,000 g. 150 μl of cell lysate was diluted by a factor of ten in ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.0], 167 mM NaCl), and protein inhibitors were added. The diluted lysate was precleared by incubation with 30 μl (60 μl for salivary glands) of Dynabeads conjugated to Protein A (Dynal) and preblocked by equilibration in 150 μl ChIP buffer containing 12 μg (24 μg for salivary glands) sonicated herring sperm DNA. The cleared lysates were then incubated with 3 μl anti-MSL1 , 2 μl anti-MSL2, or 3 μl anti-MOF (from Mitzi Kuroda, Harvard Medical School) antibodies overnight at 4 °C. The antibody complexes were precipitated by incubation with DNA-blocked Protein A Dynabeads for 1 h at 4 °C. The beads were washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM Tris-HCl [pH 8.0], 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM Tris-HCl [pH 8.0], 500 mM NaCl), once with LiCl-containing buffer (250 mM LiCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), and twice with TE Buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). The protein/DNA complexes were eluted from the antibodies by incubating them for 2 × 15 min at room temperature in 250 μl Elution buffer (1% SDS, 0.1 M NaHCO3) with rotation. NaCl was added to a final concentration of 200 mM, and protein/DNA crosslinks were reversed by heating at 65 °C for 4 h. A total of 10 μl of 0.5 M EDTA, 20 μl of 1M Tris-HCl [pH 6.5], and 1 μl of 20 mg/ml proteinase K were added before an additional incubation at 45 °C for 1 h. The DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA was then dissolved in 24 μl water. SOLiD (TM) Library Preparation - Uppsala Genome Center ; Size selection (100-150 bp + adapters 90bp < 250bp)
GEO Accession
GSM1419183