Bilateral 3mm biopsy punch defects were administered to the quadriceps muscles of (12-16-week-old) C57BL/6 mice. Mice were treated with AICAR and vehicle loaded synthetic protein nano particles (SPNPs) at the site of injury on days 1, 4, and 7 days post injury. At 8 days post injury, mice were humanely euthanized, quadriceps extracted and pooled according to treatment. After tissues were manually homogenized with scissors, they were digested into single cell suspensions for 1h at 37°C using 2 mg/ml collagenase II and 2.5 U/ml dispase II. The enzymatic reaction was stopped using 10% heat inactivated FBS with 5 mM EDTA and passed through a 70 mm strainer. Samples were centrifuged for 10 min at 300g, and resuspended in red blood cell lysis buffer for 2 min at room temperature. After washing, suspension was stained with DAPI and sorted by FACs to remove debris and dead cells (DAPI positive). Sorted cells were then labeled with 10x genomics cell multiplexing oligos (one per treatment) and then analyzed by single cell RNA sequencing. Library preparation and next-generation sequencing was carried out in the Advanced Genomics Core at the University of Michigan. Cells were loaded into the 10X Genomics chromium single cell controller for each sample and captured into nanoliter-scale gel bead-in-emulsions. cDNAs were prepared using the single cell protocol as per manufacturer's instructions.