Journal: bioRxiv

Article Title: A scalable MNase-seq framework for reproducible nucleosome profiling across pluripotent stem cell and cardiomyocyte models

doi: 10.64898/2026.06.08.731013

Figure Lengend Snippet: (A) Workflow schematic illustrating single-cell dissociation, 1% PFA fixation, 4°C storage, and MNase-mediated chromatin digestion of E14.5 mouse embryonic hearts. Scale bars: 0.5 cm (embryo), 0.5 mm (heart). Quality control pipeline for dissociated E14.5 cardiac cell suspensions, comprising cytospin preparation, immunofluorescence staining, confocal imaging, and flow cytometry. (B) Confocal images of cytospun E14.5 cardiac cells stained for cTnT (cardiomyocytes), VIMENTIN (non-cardiomyocytes), and DNA, confirming intact nuclear and membrane morphology. Scale bar: 50 µm. (C) Flow cytometric analysis of dissociated E14.5 cardiac cells, identifying ∼35% cTnT+ cardiomyocytes within the nucleated cell population, consistent with the expected composition of the E14.5 murine heart. (D) Agarose gel electrophoresis of MNase-digested chromatin from 500,000 pooled E14.5 cardiac cells (n = 3 hearts) across increasing MNase concentrations (0.5–10 U, 5 min). Lane 1: no-MNase control. (E) Comparative MNase digestion at 3 U for 2.5 min using 250,000 cardiac cells from pooled (n = 3 hearts) or individual (n = 1 heart) E14.5 embryos, demonstrating equivalent nucleosomal profiles and confirming that single embryonic hearts provide sufficient material for chromatin profiling. (F) Workflow schematic for automated adult mouse cardiomyocyte isolation using the gentleMACS Octo Dissociator with Heaters and Heart Perfusion Kit. (G) Representative confocal images of cytospun adult cardiomyocytes stained for ACTN2, MitoSpy Green, and DNA, confirming intact sarcomeric organization, mitochondrial network integrity, and high-purity cardiomyocyte recovery (n = 3). Scale bar: 100 µm. (H) Sequential flow cytometry gating of langendorf-isolated adult cardiomyocytes, showing 96.4% intact cells, 88.2% nucleated (DNA+) events, and 93.4% cTnT+ cardiomyocytes within the nucleated fraction. (I) Representative confocal images of langendorf-isolated and cytospun adult cardiomyocytes stained for ACTN2, cTnT/TNNT2, and DNA, confirming intact sarcomeric organization and high-purity cardiomyocyte recovery (n = 3). Scale bar: 100 µm. (J) Time-course MNase digestion of 250,000 adult cardiomyocyte nuclei (adjusted for ∼85% binucleation) at 3 U across 1–10 min, with clear nucleosomal ladder resolution by 2.5 min. (K) MNase concentration titration of 250,000 adult cardiomyocyte nuclei (0.5–10 U, 5 min), with optimal nucleosomal ladder resolution at 3 U.

Article Snippet: For one of the two adult cardiomyocyte isolation strategies, hearts from 2- to 4-month-old wild-type C57BL/6J male and female mice were processed using the Miltenyi Heart Dissociation Kit, mouse and rat (130-098-373, Miltenyi Biotec) on the gentleMACSTM Octo Dissociator with Heaters (130-096-427, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Single Cell, Control, Immunofluorescence, Staining, Imaging, Flow Cytometry, Membrane, Agarose Gel Electrophoresis, Isolation, Concentration Assay, Titration