Loss of skeletal muscle Bmal1 chromatin accessibility

Investigate the effects loss of skeletal muscle Bmal1 has on gastrocnemius chromatin accessibility via ATAC-seq. Overall design: Gastrocnemius muscles were collected at 6 months of age from vehicle treated and iMSBmal1KO mice. Two whole gastrocnemius muscle (~300-350mg) were diced and then homogenized using a Polytron homogenizer in homogenization buffer (Homogenization buffer (10mM HEPES, pH 7.5, 10mM MgCl2, 60mM KCl, 300mM sucrose, 0.1mM EDTA, 0.1% Triton, 1mM DTT, complete with mini protease inhibitor mix). Homogenates were spun at 300rpm for 3 minutes and supernatant containing nuclei moved to a new tube. This was repeated 3 times. The supernatant was then filtered through a 40µm cell strainer before centrifugation at 1000g for 10 minutes. The nuclei pellet was then resuspended in 1.2ml of homogenization buffer, gently triturated and 200µl loaded on top of several 2.15M sucrose cushions (10mM HEPES, pH 7.5, 10mM MgCl2, 60mM KCl, 2.15M sucrose, 0.1mM EDTA, 0.1% Triton, 1mM DTT, complete with mini protease inhibitor mix). Nuclei samples were centrifuged at 13,500g at 4°C for 1.5 hours. Samples were aspirated and washed leaving a nuclei pellet, mainly free of cytoplasmic fragments. Nuclei were quality checked using light microscopy (intact nuclei, no blebbing, free of cytoplasmic fragments) and then counted. 50,000 nuclei were used for input into a commercially available ATAC-seq kit which includes library preparation (#53150, Active Motif, Inc., CA, USA).