GSM4910656: MNase.st5.ctrl.rep2; Drosophila melanogaster; MNase-Seq
This protocol was modified from the Cut and Run protocol published by Skene and Henikoff, 2017. Embryos were smashed using a pestle in 50µl of the MNase wash buffer (20mM HEPESpH7.5, 150mM NaCl, 0.5mM Spermidine, 1x Protease inhibitor cocktail), then rinsed with 150µl of MNase wash buffer and centrifuged (600g, 3min at room temperature). Cells were wash afterwards in 500µl MNase wash buffer and resuspended in 1ml of MNase wash buffer. For eah sample, 10µl of Concanavalin A (Polyscience, 86057-3) beads were added and incubated for 15min under rotation. The beads were previously equilibrated with binding buffer (20mM HEPES pH7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2). Afterwards, the beads were resuspended in 1ml permeabilization buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.05% Digitonin, 2mM EDTA, 1x Protease inhibitor cocktail) and incubated for 2hrs under rotation. Beads were washed twice with 1ml digitonin wash Buffer (20mM HEPES pH7.5, 150mM NaCl, 0.5mM Spermidine, 0.05% Digitonin, 1xProtease inhibitor cocktail). Next, 100µl of 37°C preheated digitonin wash buffer + MNase (20U, NEB M0247S) was added to each sample. Immediately after this, 3µl of 100 mM CaCl2 were supplemented to each sample. Samples were then incubated at 37°C for 30 min. To stop the reaction, 100 µl of stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.05% digitonin, 50µg/ml RNaseA, 25µg/ml glycogen) was added. Samples were then incubated for another 30 min at 37°C. Chromatin was purified by adding 0.1%SDS and 20mg/ml of Proteinase K to each sample, followed by 1hr incubation at 50°C. DNA was afterwards purified and large fragments (>500bp) were removed using 0.5x volume of NucleoMag® NGS beads (Macherey-Nagel, 744970.50). The ChIP DNA Clean&Concentrator Kit (Zymo Research) was used to concentrate the samples according to manufacturer's instructions. Digestion efficiency was assessed by capillary electrophoresis on the Fragment Analyzer (Advanced Analytical) before preparing libraries. Libraries were prepared using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S and E6440) following the manufacturer's instructions.