title: GSM6757766: OreR, 2to3, mnase, 1; Drosophila melanogaster; MNase-Seq
library_construction_protocol: For each MNase digestion, 100 hand-sorted 2-3 h AEL Drosophila embryos were used. Nuclei were extracted by douncing in PBS with 0.1% IGEPAL CA-630. The nuclei were harvested by centrifugation and resuspended gently in MNase Digestion Buffer (PBS with 0.1% Triton X-100 and 1 mM CaCl2). MNase digestion was performed with 100 U MNase (NEB, M0247S) for 30 minutes at 37°C. The reaction was stopped with 20 mM EGTA. The nuclei were treated with 50 µg/ml RNase A (Thermo Scientific, EN0531) for 1 hour at 37 °C and 1000 rpm, and subsequently incubated overnight at 65 °C and 1000 rpm with 200 µg/ml Proteinase K (Invitrogen, 100005393) and 0.5% SDS for reverse cross-linking. DNA was extracted using phenol-chloroform (VWR, K169). MNase-seq libraries were constructed from 10 ng purified DNA using the High Throughput Library Prep Kit from KAPA Biosystems (KK8234) according to the manufacturer's instructions. MNase-seq for nucleosome occupancy using standard Illumina protocols.