GSM7439446
After 10 days exposure to IL-1β and or butyrate, the islets (500-600 per condition) were washed in PBS. Cross-linking was performed in 0.5 % formaldehyde/PBS solution at room temperature for 7.5 min and quenched with 0.125 M glycine for 5 min. The islets were washed with PBS before resuspension in 1 mL of IP buffer (67 mM TrisHCl (pH 8), 100 mM NaCl, 5 mM EDTA (pH 8), 0.2 % NaN3, 0.33 % SDS, 1.67 % Triton X100, supplemented with protease inhibitor). Homogenization was performed by passing the islets through a 25 g needle. Chromatin was sonicated to an average length of 200-400 bp (~30 cycles, high intensity on Diagenode, Biorupter). Chromatin was cleared by centrifugation at 20,000 g for 30 min. For each ChIP, 700 ng DNA was incubated with 4 μg H3K27ac antibody (cat# Ab4729, Abcam) overnight rotating at 4 °C. Chromatin-antibody complexes were captured using Protein G Sepharose 4 Fast Flow beads (GE healthcare, Broendby, Denmark) for 4 hours followed by three washes with low-salt buffer (20 mM TrisHCl (pH 8), 2 mM EDTA, 1 % Triton X-100, 0.1 % SDS, 150 mM NaCl) and a final wash with high-salt buffer (20 mM TrisHCl (pH 8), 2 mM EDTA, 1% Triton X-100, 0.1 % SDS, 500 mM NaCl). Chromatin was decrosslinked in 1 % SDS and 0.1 M NaHCO3 for 6 hours at 65 °C. DNA was purified using Qiagen MinElute PCR purification kit (Qiagen, Aarhus, Denmark) and the concentration was determined using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) DNA libraries were prepared with 7-14 ng of DNA usingthe NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs, Hitchin, UK) and Unique Dual Index UMI Adaptors DNA set 1 (New England Biolabs) for Illumina sequencing according to the manufacturer's protocol. For each library preparation, the optimal number of PCR cycles was determined by running a real-time PCR reaction using Brilliant III Ultra-fast SYBR Green QPCR Master Mix (Agilent, Santa Clara, CA, USA) and a C1000 Thermal cycler (Bio-Rad). The quality and the quantity of the libraries were checked on Bioanalyzer 2100 instrument (High Sensitivity DNA kit, Agilent) and DNA quantified by Qubit (Thermo Fisher Scientific) prior to sequencing.