A Scalable MNase-seq Framework for Reproducible Nucleosome Profiling across Pluripotent Stem Cell and Cardiomyocyte...

Micrococcal nuclease (MNase) digestion is widely used to profile chromatin accessibility and nucleosome footprinting. However, its application is often limited by sensitivity to reaction conditions, high cell input requirements, and the lack of standardized protocols across cell types. Here, we developed a robust MNase workflow encompassing optimization of buffer composition, DNA purification chemistry, fixation and decrosslinking parameters, cell input scalability, and an in-house yeast spike-in for quantitative normalization. We validated this unified framework across human induced pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes at multiple differentiation stages, as well as in primary isolated murine embryonic cardiac cells and adult mouse cardiomyocytes, and demonstrated comparable digestion efficiencies and kinetics despite marked differences in cellular architecture and chromatin organization. Robust nucleosomal digestion further extended to a panel of 6 unrelated human and mouse immortalized cell lines. Genome-wide MNase-seq in hiPSCs, combined with the nucMACC bioinformatic pipeline, resolved enzyme concentration-dependent nucleosomal occupancy and precise nucleosome positioning at pluripotency-related regulatory elements. This modular, end-to-end, and scalable workflow provides a standardized platform for reproducible MNase-based chromatin digestion across diverse in vitro and in vivo models, and for genome-wide nucleosome profiling in pluripotent stem cells.