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

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Micrococcal nuclease (MNase) digestion is crucial for chromatin accessibility and nucleosome footprinting studies.
• Current MNase protocols face limitations including sensitivity to reaction conditions, high cell input needs, and lack of standardization.

Purpose of the Study:

• To develop a robust, standardized, and scalable MNase workflow for reproducible chromatin digestion and nucleosome profiling.
• To overcome limitations of existing MNase methods for diverse cell types and experimental models.

Main Methods:

• Optimized buffer composition, DNA purification, fixation/decrosslinking, and cell input scalability.
• Incorporated an in-house yeast spike-in for quantitative normalization.
• Validated the workflow across human induced pluripotent stem cells (hiPSCs), cardiomyocytes, and murine cardiac cells using MNase-seq and the nucMACC pipeline.

Main Results:

• Demonstrated comparable MNase digestion efficiencies and kinetics across diverse cell types, including hiPSCs, cardiomyocytes, and murine cells.
• Successfully applied the workflow to 6 immortalized human and mouse cell lines.
• Resolved enzyme concentration-dependent nucleosomal occupancy and precise nucleosome positioning in hiPSCs.

Conclusions:

• The developed workflow provides a standardized, modular, end-to-end, and scalable platform for reproducible MNase-based chromatin digestion.
• Enables genome-wide nucleosome profiling in various in vitro and in vivo models, particularly in pluripotent stem cells.