Optimized RNA and Protein Isolation from Muscle Cells and Tissues

For RNA isolation, myoblasts and myotubes were collected in 500 μL Tri-Reagent (Sigma-Aldrich) and stored at −20°C until processing. For protein isolation, cells were collected in 200 μL of RIPA buffer (Millipore) containing PhosStop phosphatase inhibitor cocktail tablets (Roche) and cOmplete Mini, EDTA-free protease inhibitor cocktail tablets (Roche) on ice. Samples were then incubated on ice for 30 min with vortexing to ensure lysis and then centrifuged for 10 min at 16,000 x g at 4°C. Supernatants were collected and stored at −20°C.
Mice were euthanized and muscles were dissected and snap-frozen in liquid nitrogen and stored at −80°C. Tissue was lyophilized for 8 h and pulverized with ceramic beads on dry ice using a Bertin Technologies Percellys Evolution Tissue Homogenizer (Molnar et al., 2021 (link)). Pulverized tissues were then homogenized on ice with a Polytron PT1200-E in either 500 μL Tri-Reagent or 100 μL RIPA buffer supplemented with protease and phosphatase inhibitor cocktail tablets (Roche). Homogenized tissues in Tri-Reagent were stored at −20°C. Homogenized tissues in RIPA buffer were centrifuged for 10 min at 16,000 x g at 4°C, and the supernatant was stored at −20°C.