GSM1319842: sd_ip; Drosophila melanogaster; ChIP-Seq
Chromatin immunoprecipitation from eye discs was performed using a modified protocol from (Gaertner et al., 2012). First, third instar larvae were dissected in PBS (pH 7.4) such that only eye discs and brain remained attached to the mouth hooks. The dissected material was subsequently fixed in 1ml fixation buffer (50 mM HEPES, pH 7.5; 1 mM EDTA; 0.5 mM EGTA; 100 mM NaCl; 2% formaldehyde) for 30 minutes at room temperature. After four washes (PBS, pH 7.4; 0.1% Triton X-100; 0.1% Tween-20), eye discs were hand-dissected and combined into pools of 200 discs in 300 µL buffer A2 (15 mM HEPES, pH 7.5; 140 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1 % SDS; 0.5 % N-lauroylsarcosine; 1x Roche complete protease inhibitor cocktail, cat# 5056489001). Sonication was performed in a Bioruptor sonicator for 30 minutes (30 sec on/off cycle at the "high" setting). Following centrifugation (16,000 x g; 10 minutes at 4°C), the supernatant containing soluble chromatin was transferred to fresh tubes, and 50 µL was set aside as whole cell extract (WCE; input). Per ChIP, 10 ug antibodies were added to 450 µl chromatin (corresponding to approximately 300 discs) and incubated overnight at 4 °C with rotation. We used the following antibodies: anti-Pol II (Covance 8WG16, cat# MMS-126R; mouse monoclonal antibody), anti-Sd, and anti-Yki. Immunocomplexes were purified by adding 50 µL pre-washed Dynabeads coated with protein A/protein G (Life Technologies, cat#10002D and 10004D) for four hours, rotating at 4 °C. The beads were washed three times in RIPA buffer (50 mM HEPES, pH 7.5; 1 mM EDTA; 0.7% sodium deoxycholate; 1% NP-40; 500 mM LiCl) and once in TE. Immunoprecipitated DNA was eluted twice in 75 µL elution buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS) at 65°C to maximize yields. Crosslinks of ChIP and WCE DNA were reversed over night at 65°C. DNA was purified by RNAse A (Sigma, cat# R6513; [0.2 µg/µl]; 1 h at 37 °C) and proteinase K (Life Technologies, cat# AM2546; [0.2 µg/µl]; 2 h at 55 °C) treatment followed by phenol/phenol-chloroform-isoamylalcohol extractions and ethanol precipitation. The precipitated DNA was resuspended in 30 ul 10 mM Tris buffer (pH 8). For ChIP-seq library preparation, 30 ng ChIP DNA and 100ng WCE DNA were used to construct ChIP-Seq libraries with the NEBNext ChIP-seq Library Prep Master Mix Set (cat# E6200L) and the NEBNext Multiplex Oligos (cat# E7335S and E7500S) for Illumina, following the manufacturer's instructions. Concentration and size distribution of the libraries were assessed on an Agilent 2100 Bioanalyzer (High Sensitivity DNA assay chip).