B6 cg tg hist1h2bb egfp 1pa j mice

We obtained B6.Cg-Tg(HIST1H2BB/EGFP)1Pa/J mice from Jackson Laboratory (#006069). All animal procedures were performed following Institutional Animal Care & Use Committee (IACUC) approved protocols at University of Colorado Boulder (Protocol number: 1507 and 2628). Nuclei of the cells from homozygous mice bred from this colony display a strong fluorescence at 488 nm caused by the green fluorescence tag attached to its histone H2B. Because histone H2B is ubiquitous in all nucleosomes in the chromatin, fluorescence intensity of H2B linearly correlates with the chromatin density in the nucleus. The fluorescent labeling of chromatin through this green fluorescent tag is routinely used in labs ^{[55 (link)]} to study chromatin dynamics in live cells and we showed that the use of this tag is equivalent to other live chromatin stains such as Hoechst or DRAQ5 for the visualization of chromatin ^{[24 (link)]}. Cardiomyocytes were isolated from embryonic mice hearts 18.5 days post conception by incubation of the tissue in 0.125% trypsin/EDTA overnight followed by 10 min digestion in residual trypsin under the application of medium maintained at 37°C. After isolation, cells were cultured on PDMS substrates. Cardiomyocytes were cultured in Advanced DMEM/F12 containing 10% FBS at 37°C with 5% CO₂ until the imaging experiment was carried out.

B6.Cg-Tg (HIST1H2BB/EGFP) 1Pa/J mice (Stock No: 006069), referred to as H2b-eGFP mice, were obtained from Jackson Laboratory. All animal procedures were performed following Institutional Animal Care and Use Committee approval. Embryonic mice hearts were harvested 18.5 days post conception. Hearts were minced and incubated in a digestive mix for 30 min at 37°C. Digestive mix contained 2 mg/ml Papain (P4762, Sigma), 500 µg/ml Liberase TM (05401119001, Roche), 5 mM L-Cysteine (C6852, Sigma) and 10 µg/ml DNase-I (D4263, Sigma). Cardiac cells were isolated through gentle trituration using a 1 ml pipette and cells were cultured on prepared substrates in DMEM-F12 Advanced (Gibco) containing 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco) and 25 mM HEPES (Gibco) at a density of 50,000 cells/cm². Samples were incubated at 37°C and 5% CO₂⁷⁷ . Cultures were washed in medium 24 hours after plating to remove any debris. Half medium changes were performed on Day 2 in culture.
For H3K9M and H3WT studies, male mice carrying the transgene were bred to C57BL/6 (Jackson Laboratory) females. Embryonic hearts were harvested on day 18.5 post conception. Tissue digestion and culture conditions were maintained the same as stated for the H2b-eGFP cardiac cultures. On Day 0, 1, and 2, cells were induced with 2µg/ml doxycycline (Sigma).