This protocol describes isolation of nuclei from 10 week old mouse tibialis anterior from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using the Parse Biosciences protocol. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish.