library_construction_protocol: 10-20 fly ovaries were dissected in ice cold PBS per sample.  Ovaries were digested in 250 µl of dissociation buffer (0.5% Trypsin and 2.5 mg/ml Collagenase A in PBS) for an hour with shaking at 800 rpm at 30 °C.  The digestion was stopped with 250 µl of Schneider medium containing 10% FBS.  Cell suspensions were filtered through 40 µm strainers, pelleted and washed three times with Wash buffer 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.