MNase-seq of Drosophila S2 cells

We report the MNase-diestion coupled to Next Generation Sequencing of Wild type Drosophila S2 cells or S2 cells over-expressing polycomb protein PH Cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature, tumbling end over end. Crosslinking was quenched by 1M glycine (from 2.5M pH 7.9 stock) and cells were tumbled for 10min at RT. Cells were resuspended in cold PBS (+PI), pelleted through a sucrose cushion (20% sucrose in PBS), resuspended in PBS, and flash frozen in liquid nitrogen at 107 cells per tube and stored at -80oC. For MNase digestion, the cell pellet was resuspended in PBS with 0.1% Triton-X 100 (PBS-TX). Digestion of 10^6 cells per titration point took place in a volume of 400uL PBS-TX supplemented with 1mM CaCl2. Either 1.5 U, 6.25 U, 25 U, 100 U or 400 U of MNase (Worthington Biochemical) were added to pre-warmed cells and incubated at 37 oC for 3 minutes. Digestion was stopped by moving samples to ice and adding of 10 uL of 250 mM EDTA, 250 mM EGTA, and 10 uL of 20% SDS. For DNA cleanup, the digestion products were incubated with RNase (Roche) for 30 minutes at 37 oC, with proteinase K (2.5 ul of 20 mg/ml) (Roche) for 60 minutes at 55 oC, and incubated at 65 oC for overnight to reverse crosslinks. Following phenol-chloroform extraction and ethanol precipitation, the purified DNA was used input into the library preparation