GSM7884466: S2-siEz.H3K9me3.3 [CUT&RUN]; Drosophila melanogaster; OTHER
1 million cells per sample were harvested and washed three times with Wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine supplemented with protease inhibitors) and resuspended in 1 ml of Wash buffer.   Following sample preparation, CUT&RUN was performed according to instructions in CUT&RUN protocol V.3 with some modifications ).  10 µl of activated Concanavalin A-coated magnetic beads (Bangs Laboratories) were added to each sample and rotated for 10 min at RT.  Bead-bound cells were incubated with 5 µl of antibody (Table S3) and 95 µl of antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin) overnight at 4 °C.  Bead-bound cells were washed twice with Dig-wash buffer (0.05% digitonin in Wash buffer).  Bead-bound cells were resuspended in Dig-wash buffer containing 1x CUTANA™ pAG-MNase (Epicypher) and rotated for 1 hr at RT.  Following pAG-MNase binding, bead-bound cells were washed twice with Dig-wash buffer and resuspended in 100 µl Dig-wash buffer.  Chromatin digestion was performed on ice for 30 min by adding 2 μL 100  CaCl2 mM.  Digestion was stopped by the addition of 2x STOP Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 µg/ml RNase A (Thermo Fisher Scientific), 50 µg/ml glycogen) and samples were incubated at 37 °C for 30 min to release DNA fragments into the solution.  After centrifugation, 0.1% SDS and 0.2 μg/μl Proteinase K were added to the supernatant and samples were incubated for 1 hour at 50 °C.  DNA was extracted using Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (Sigma) according to the manufacturer's instructions. Libraries were prepared following the manufacturer's instructions with NEBNext Ultra II DNA Library Prep Kit for Illumina.