GSM1644833: ACF1-Rb1 acf1 rep1; Drosophila melanogaster; ChIP-Seq
Un-staged embryos were dechorinated using 120 ml final, 1:5 diluted, sodium hypochloride for 3 min. The embryos were thoroughly washed and fixed in the fixing solution [30 ml n-Heptane solution and 10 ml fixing buffer containing 0.1 M NaCl, 0.05 M HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 3.7% Formaldehyde for 15 min at 16-18°C on a rotating wheel. Fixation was then quenched by adding 125 mM, final, Glycine to the tube. The embryos were subsequently washed using PBS [+ 0.01% Triton-X100] for 10 min and stored at -80°C until further use. For nuclei isolation, embryos were slowly thawed and dounced using glass homogenizer A and B (20 strokes each) in ice-cold NX-I buffer [15 mM HEPES pH 7.6, 10 mM KCl, 2 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 350 mM sucrose, 1 mM DTT, 0.2 mM PMSF, Protease inhibitors Leupeptin, Pepstatin and Aprotinin (10 µg/ml)]. The lysate was filtered through Miracloth and nuclei were spun down at 3500 rpm, 10 min, 4°C. The pellet (brown) was washed in NX-I buffer. Finally, the nuclei were resuspended in RIPA [1% Triton X-100, 0.1% Sodium deoxycholate, 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS, 1 mM PMSF] and washed 3 times. Nuclei were then counted and aliquoted to ~109 nuclei/ml. Fractions can be stored at -80°C until shearing. For shearing and ChIP, thawed nuclei were adjusted to ~2x108 in RIPA and sheared in Covaris S220 system (Covaris Inc. MA, USA) at power 110 Watts, duty factor 20%, and cycles per burst 200 for 25 min. Chromatin was pre-cleared using protein A+G beads (1:1) mix for 1h, 4°C. 200 µl of chromatin was taken for chromatin immunoprecipitation in 500 µl reaction. The ChIP antibodies are all custom antibodies produced in our lab.  4 µl of antibodies (Rb1-ACF1, Rb2-ACF1, Rb-RSF1) were incubated per reaction overnight at 4°C. RIPA equilibrated protein A+G (1:1) mix was then added to pull down the immune-complexes for 3h, 4°C. For Rat monoclonal antibody, the chromatin immune-precipitation step was performed using presorbed protein G beads, in an excess of antibody, for 3hr at 4°C. Beads were washed subsequently 5 times in 1 ml RIPA buffer. Residual RNA was digested by RNase (10 µg) at 37°C for 20 min. Subsequent protein digestion (25 µg Proteinase K) and reversal of cross-linking were performed together at 68°C for 2 hrs. DNA was purified using GenElute™ PCR Clean-Up Kit (Sigma, Cat.no NA1020). ChIP DNA was quantified using Qubit® dsDNA HS Assay Kit (Life technologies, Cat.no.Q32851) and sequencing libraries were prepared using MicroPlex Library Preparation™ kit (Diagenode, Cat. No. C05010011) by taking 2 ng of starting material whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).