GSM4474725: Input_VMH; Mus musculus; ChIP-Seq
ChIP was performed with anti-HA magnetic beads (Pierce 888367). 10 independents hypothalamic nuclei punches were pooled together per biological replicate. Hypothalamic nuclei punches were directly thawed in cross-linking solution during 20min at room temperature (1% formaldehyde in 1X PBS) , quenched with 0.125M of glycine during 5min and washed three times with 1XPBS at 4°C. Cross-linked hypothalamic nuclei punches were re-suspended un ChIP dilution buffer (50 mM HEPES pH7.5, 155 mM NaCl, 1 mM EDTA, 1.1% Triton X-100, 0.11% Sodium-deoxycholate, 0.1% SDS) on ice and re-suspend during chromatin fragmentation at 4°C (Fischer Scientific, FB705 Sonic Dismembrator : three cycles at 10% amplitude, three cycles of at 15% amplitude). Lipid-cleared samples were resuspended in 1mL with ChIP dilution buffer and input was saved before incubation with BSA blocked HA magnetic beads (Pierce, 888367) overnight at 4°C with rotation. Immunoprecipitation were washed on rotation at 4°C with ChIP dilution buffer twice (1min and 5min), ChIP dilution buffer with 500mM NaCl (5min), ChIP wash buffer (5min, 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 (5min, 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 before precipitation by NaCl/EtOH overnight at -20°C with 20ug of glycogen carrier (Thermo Fisher Scientic, AM9515). ChIP DNA was prepared for sequencing according to the amplification protocol provided by Illumina