GSM2849524: MNase-Seq Gal4DBD; N1(UAS-LacZ-mini-white) replicate 2; Drosophila melanogaster; MNase-Seq
Eggs laid during a 6 hr period were collected and aged for a further two hours at 25°C. Subsequently, one gram embryos were dechorinated in 120 ml 1:5 diluted sodium hypochloride (VWR, Cat.no. 301696S) for 3 min, thoroughly washed and fixed in 10 ml fixing solution [0.1 M NaCl, 0.05 M HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1.8% Formaldehyde (FA; Sigma, Cat. No F1635) added to 30 ml n-Heptane (VWR, Cat. No. 8.22332.1000)] for 15 min at 16-18°C on a rotating wheel. Fixation was quenched by adding 125 mM glycine. The embryos were subsequently washed with PBS (including 0.01% Triton-X100) for 10 min and stored at -80°C. For nuclei isolation, embryos were slowly thawed and dounced using a glass homogenizer (Schubert, Cat.no. 9164693) with 20 strokes each of the A and B pestles 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 pelleted at 3,500 rpm, 10 min at 4°C. For MNase digestion, the nuclei were resuspended in the MNase digestion buffer. About 80 mg of nuclei were digested with 13 units of MNase (Sigma, Cat.no N5386) for 15 min, 37°C while shaking at 500 rpm. The reaction was stopped by adding 20 µl of 0.5 M EDTA, pH 8.0 and the tubes were quickly transferred to ice for 1 min. Nuclei were spun at 12,500 rpm, 10 min at 4°C. The supernatants containing most of the DNA were collected and the buffer composition was aDJ.gzusted to RIPA [1% Triton X-100, 0.1% Na deoxycholate, 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS, 1mM PMSF]. The digestion predominantly yielded mononucleosomes. Recovered DNA was quantified using the Qubit® dsDNA HS Assay Kit (Life Technologies, Cat.no.Q32851) and sequencing libraries were prepared using the MicroPlex Library Preparation kit (Diagenode, Cat. No. C05010011) starting with 2 ng DNA whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).