Chromatin Immunoprecipitation–qPCR

Chromatin immunoprecipitation (ChIP) experiments were performed in RL14 cells using the ab‐500 ChIP kit (Abcam, Cambridge, MA), following the manufacturer's protocol. Briefly, the cells were cross‐linked in 1% formaldehyde (Sigma‐Aldrich, St Louis, MO) for 10 minutes at room temperature and cross‐linking was stopped with 0.125 mol/L of glycine. Chromatin was fragmented by sonication using Fisher Scientific 50 Sonic Dismembrator Cell Disruptor (Fisher Scientific, Pittsburgh, PA). For immunoprecipitation, a 100‐μL aliquot of sonicated chromatin was incubated at 4°C overnight with ChIP grade anti‐MEF2C antibody (Abcam, Cambridge, MA). The antibody‐chromatin complex was then incubated with protein A beads at 4°C for 1 hour. The beads were washed, and 100 μL of DNA purifying slurry was added, mixed well, and incubated at 98°C for 10 minutes. After leaving the reaction at room temperature for 20 minutes, 1 μL of proteinase K was added and incubated at 55°C for 30 minutes. After incubation at 98°C for 10 minutes, DNA purifying slurry was pelleted by centrifugation. Supernatant was collected for qPCR analysis. qPCR was conducted using gene‐specific primers (Table), Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA), and 7500 Fast Real‐Time PCR System (Applied Biosystems). The enrichment of SCN5A DNA fragment was normalized with that of the input sample. We used the percentage input value method to calculate the relative abundance of SCN5A fragments in the ChIP assay. Fold enrichment was determined by dividing the enrichment of the immunoprecipitated chromatin with MEF2C antibody by that with control IgG.