Optimized Low-Input ATAC-Seq Protocol for Freshly Sorted Muscle Stem Cells

Skeletal muscle stem cells (MuSCs) are essential for tissue regeneration and repair, yet their scarcity has historically limited routine application of chromatin-level assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) provides a powerful means to define the chromatin state and study cellular regulatory landscapes, but standard protocols require cell numbers far exceeding those that can be freshly sorted from muscle, particularly under conditions such as aging, disease, and various genetic models. Here, we present an optimized, low-input ATAC-seq workflow specifically tailored for analysis of MuSCs freshly isolated from skeletal muscle. This method integrates high-purity isolation of MuSCs by fluorescence-activated cell sorting (FACS), refined nuclei purification, and a high-efficiency transposition assay that can be applied to the analysis of chromatin using as few as 5000 cells. We describe critical steps for minimizing technical noise, including optimized mitochondrial depletion, micro-volume tagmentation, low-cycle ATAC-seq library amplification, and quantitative quality-control metrics. The workflow is compatible with FACS-purified MuSCs from young and aged mice, injury models, and genetic knockout models, enabling direct comparison of chromatin accessibility across biological conditions where cell numbers are limiting.