50 million S2 cells were harvested and washed with PBS. The cell was resuspended in 1ml of crosslinking solution (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), 37% formaldehyde was added to a final concentration of 1.8%. The tube was rotated at room temperature for 10 minutes and the reaction was quenched with 2.5 M glycine at a final concentration of 125 mM. The cells were spun down immediately after addition of glycine, and the solution was replaced with fresh PBS with 125 mM glycine. For NSL1 and NURF301 MNase-ChIP-seq, we used MNase for chromatin shearing. Cells were washed as above described and suspended in MNase digestion buffer. A small aliquot of the samples is used to determine the optimal MNase concentration (20-200 U/ml tested) to obtain the desired fragment size. Digestion is performed in room temperature for 20 minutes and finally quenched by addition of 1 mM EDTA and 2X RIPA buffer.
GEO Accession
GSM3344581