GSM3319507: Ecc15 infection, input DNA; Drosophila melanogaster; ChIP-Seq
For each condition, 100 guts were dissected from w1118 adult female flies. Flies were kept in liquid nitrogen and batches of 20  Guts were dissected on ice, frozen in liquid nitrogen and stored at -80°C. On the day of the experiments, guts were homogenized in NE Buffer (15mM HEPES, 10mM KCl, 0.1mM EDTA, 0.5 mM EGTA, 350mM Sucrose, 0.1% Tween-20, 5mM MgCl2, 1mM DTT, 1mM PMSF, protease inhibitor tablet) supplemented with 1% formaldehyde using a douncer and pestle. After 10 minutes, crosslinking was quenched by the addition of Glycine for a final concentration of 0.125M.  Samples were cleared by centrifuging for 4 min at 4000 rpm and 4°C. Samples were washed twice with ice-cold NE buffer and twice with ice-cold RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 0.5% Na-deoxycholate, 0.5mM DTT, 0.1% SDS, 1% NP-40, protease inhibitor tablet). Finally, samples were resuspended in 130 μl RIPA buffer and sonicated in Covaris E-220 (30 seconds, Intensity: 175, Cycles per burst 200, Duty 20%, Water level: 10). Samples were then cleared by centrifugation for 10 min, 4°C max speed. At this point, 1% of the total volume was separated as input and stored at 4°C, then, the remaining amount was diluted 1:5 in IP Dilution buffer (2.8 ml H2O, 3 μl 10% SDS, 7.2 μl 0.5M EDTA, 33 μl Triton X-100, 50.1 μl Tris-HCl pH 8.1, 100.2 μl 5M NaCl). We then added 1 μg of antibody (Abcam ab5408) and incubated the sample overnight at 4°C on a rotating platform. The next day, the sample was transferred to a tube containing 50 μl of magnetic beads (M-280 Sheep Anti-Mouse IgG) blocked overnight in Beads Blocking Buffer (8.77ml PBS 1x, 1 ml BSA 1%, 10 μl Triton X-100, 220 μl 45% Fish Gelatin) and the mixture was incubated for 2 hours at 4°C on a magnetic platform. Using a magnetic racks, beads were washed once with Low Salt Buffer (20mM Tris-HCl pH 8.1, 150 mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100), twice with High Salt Buffer (20mM Tris-HCl pH 8.1, 500 mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100), LiCl Buffer (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1mM EDTA, 1% NP-40, 1% NA-deoxycholate) and TE-NaCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). In between each wash, beads were incubated 10 min at 4°C on a rotating platform. After the last wash, beads are resuspended in 500 μl of Elution Buffer (3.24 mL H2O, 50 μl Tris-HCl pH 7.5 1M, 10 μl EDTA 0.5M, 1 mL NaHCO3 0.5M, 500 μl 10% SDS, 200 μl NaCl 5M) and the input sample was supplemented with the same amount. From then on, both the input and the IP were treated similarly. We first incubated them at 37°C for 30 min with 900 rpm shaking in the presence of 7.5 μl RNAse A 20 mg/ml. We then added 10 μl of Proteinase K and incubated the sample at 55°C overnight. The next day, we added 10 μl of Proteinase K and incubated for 1h at 45°C.  Samples were then spin down for 5 min at room temperature and 2000 rpm, finally, we used 500 μl of samples as starting material for Qiagen PCR purification kit, following the manufacturer instructions. We eluted the the IP and the input in 30 μl. We used the Qubit dsDNA HS kit to measure the DNA load. 10 ng of DNA were transferred to a low binding tube and completed to 55.5 μl with H2O. We added 3 μl of NEBNext Ultra End Repair / dA-Tailing Module Enzyme mix and 6.5 μl of Reaction buffer and incubated each tube at 20°C for 30 min, then 65°C for 30 min. The product of the reaction was purified using the Qiagen MinElute PCR Purification Kit, elution was made in 12.5 μl of Elution Buffer. For each tube, an adapter with a different barcode was selected. We used the DNA Quick ligase kit, using 15 μl of 2x buffer, 1.5 μl of DNA quick ligase and 1 μl of adapter hybrid primer. Mixture was incubated at 22°C for 30 min. The reaction was purified using the Qiagen MinElute PCR Purification Kit, elution was made in 50 μl of Elution Buffer. Samples were purified using AMPure beads in a 1:1 ratio, washed twice with 80% EtOH and resuspended in 20 μl of Elution Buffer. Using 1 μl, we perfrom a qPCR using the KAPA SYBR green kit 50 μl total volume to determine the number of cycle for each samples. We then amplify each sample by PCR using the KAPA master mix. We then perform a size selection using AMPure beads, first using a 0.6.1 ratio and excluding the bound fraction followed by a 1:1 ratio selection, washing twice with 80% EtOH and resuspending in 20 μl Elution Buffer. We used in 1 μl to measure the DNA load with Qubit dsDNA HS assay and 1 μl to assess the fragment profile using the Agilent Bio-analyzer DNA 12000 kit.