GSM3781205: E11E_MNase_INP; Drosophila melanogaster; ChIP-Seq
1 g embryos were washed in 50 mL PBSTx-0.01% (PBS/0.01% Triton X-100 ) and resuspended in 10 mL fixation solution (50 mM Hepes pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA)/3.7% formaldehyde (Merck, Cat. No. 1040031000). After addition of 30 mL n-heptane, embryos were rigorously shaken for 1 min and incubated on a rotating wheel at room temperature for 13.5 min. Following a spin at 2000 g for 1 min, cross-linking was halted by addition of 50 mL PBSTx-0.01%/125 mM Glycine. After two washes with PBSTx-0.01% for 10 min each, embryos were flash-frozed and stored at -80°C until further use.  To process, frozen embryos were resuspended in 10 mL Homogenization Buffer (15 mM Hepes pH 7.6, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 350 mM sucrose, 1 mM DTT, 0.2 mM PMSF, Roche cOmplete Protease inhibitor without EDTA) and dounced 20 times with a loose pestle and 20 times with a tight pestle before being spun down at 170 g for 10 min at 4°C. The pellet consisting of nuclei was then resuspended in 4 mL RIPA Buffer (25 mM Hepes-NaOH pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM PMSF, Roche cOmplete Protease inhibitor without EDTA). Fragmentation of chromatin was done either by treatment with the MNase enzyme (Sigma, Cat. No. N5386) or sonication shearing using the Covaris S220 instrument. To obtain similar digestion degree, stage 5-8 embryo were digested using 0.9 units MNase/g embryo and stage 13-15 embryo were digested using 2.9 units MNase/g embryo at 37°C for 30 min, shaking. Reaction was stopped by the addition of EDTA to a final concentration of 10 mM. Additional mechanical shearing was done by passing lysate through a 27G needle 15 times. Alternatively, sonication was performed at 100 W Peak Power, 20% Duty Factor, 200 Cycles/Burst for 20 min. Thereafter, soluble chromatin was retrieved by centrifugation at 15000 g for 15 min at 4°C. Chromatin fragment size distribution was evaluated on a Bioanalyzer (Agilent). 1-2 µg soluble chromatin was used as input for each ChIP. In short, pre-cleared soluble chromatin was incubated with antibody in RIPA buffer overnight and retrieved by incubation with 50% slurry mix of protein A+G (1:1) sepharose beads. Reversal of cross-linking was done by an overnight incubation, shaking at 65°C, followed by an incubation with 1 µg RNase for 30 min at 37°C and with 0.1% SDS/1 µg Proteinase K for 1.5 hrs at 55°C. DNA was then purified by phenol-chloroform extraction using MaXtract tube (Qiagen, Cat. No. 129046). Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New Englands Biolab, E7654).
GEO Accession
GSM3781205