Ten independent hypothalamic nuclei punches were pooled together. Hypothalamic nuclei punches were directly thawed in cross-linking solution for 20 minutes at room temperature (1% formaldehyde in 1× PBS), quenched with 0.125 M glycine for 5 minutes, and washed 3 times with 1× PBS at 4°C. Cross-linked hypothalamic nuclei punches were resuspended in ChIP dilution buffer (50 mM HEPES pH 7.5, 155 mM NaCl, 1 mM EDTA, 1.1% Triton X-100, 0.11% sodium deoxycholate, 0.1% SDS) on ice and resuspended during chromatin fragmentation with a Sonic Dismembrator (Thermo Fischer Scientific, FB705) at 4°C: 3 cycles at 10% amplitude, 3 cycles at 15% amplitude. Lipid-cleared samples were resuspended in 1 mL of ChIP dilution buffer and input was saved before incubation with BSA-blocked HA magnetic beads (Pierce, 888367) overnight at 4°C with rotation. Immunoprecipitates were washed with rotation at 4°C with ChIP dilution buffer twice (1 and 5 minutes each), ChIP dilution buffer with 500 mM NaCl (5 minutes), ChIP wash buffer (5 minutes; 10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and Tris-EDTA buffer (5 minutes; 10 mM Tris-HCl pH 8.0, 1 mM EDTA). Cross-linking was reversed overnight at 65°C in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and DNA was isolated using phenol/chloroform and precipitated by NaCl/EtOH overnight at –80°C with 20 μg of glycogen carrier (Thermo Fisher Scientific, AM9515).

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