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<p id="__p11">Testicular germ cell enrichment was performed on 14-day postpartum male mice as previously reported (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib6" id="__tag_751284714" rid="bib6" role="button">Baker <em>et al.</em> 2014</a>). This cell preparation removes somatic Sertoli and Leydig cells and results in >90% enrichment of germ cells, of which nearly 50% are spermatogonia (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib8" id="__tag_751284677" rid="bib8" role="button">Ball <em>et al.</em> 2016</a>).</p>
<p id="__p12">Individual low-passage mouse ESCs were derived using protocols outlined in <a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib14" id="__tag_751284767" rid="bib14" role="button">Czechanski <em>et al.</em> (2014)</a>. Briefly, 6- to 8-week-old females are mated to stud males and checked each morning for plugs. Pregnant females are euthanized on embryonic day 3.5 and the uterine horn is flushed to remove embryos. Embryos are visualized under a dissecting microscope and blastocysts are transferred to 2i (2i:CHIR99021 and PD0325901) (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib72" id="__tag_751284764" rid="bib72" role="button">Ying <em>et al.</em> 2008</a>) serum-free media for outgrowth of the inner cell mass. Blastocysts are allowed to hatch and attach to a mouse embryonic fibroblast (MEF) feeder layer, and the resulting outgrowth is monitored daily and fed for 8–11 days. The emergent ESCs are disaggregated and passaged onto new MEF feeders. Cultures during this time are closely monitored for unusually rapid growth (potentially indicating karyotypic instability), signs of deterioration including vacuolated cytoplasm, detachment of cells from colonies and debris, and possible signs of contamination. Successful ESC cultures were maintained on MEF feeders in serum containing 2i media supplemented with Leukemia inhibiting factor (2i/LIF) (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib28" id="__tag_751284752" rid="bib28" role="button">Kiyonari <em>et al.</em> 2010</a>) to maintain high levels of NANOG expression, which indicates ground-state pluripotency (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib72" id="__tag_751284746" rid="bib72" role="button">Ying <em>et al.</em> 2008</a>; <a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib14" id="__tag_751284768" rid="bib14" role="button">Czechanski <em>et al.</em> 2014</a>). Prior to preparing for chromatin isolation, mouse ESCs were enzymatically disassociated using trypsin and MEFs were removed by serial plating on gelatin-coated plates to which MEFs adsorb preferentially; for this, ESCs and MEFs are incubated in 2i/LIF media on fresh plates for 15 min to allow the larger MEFs to quickly attach to the plates. ESCs are aspirated and the plating procedure repeated once to further remove MEFs. ESCs were collected by centrifugation, resuspended in PBS, and cross-linked using formaldehyde.</p>
<p id="__p13">For hepatocyte isolation and purification, livers from 8-week-old female mice were perfused using a modified EGTA–collagenase perfusion protocol (<a aria-expanded="false" aria-haspopup="true" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404261/#bib44" id="__tag_751284772" rid="bib44" role="button">Neufeld 1997</a>). All perfusions and hepatocyte purifications were done at the same time of the day to avoid possible circadian effects on any studied parameter. EGTA buffer was used to flush the blood out of the liver and start to digest the desmosomes connecting the liver cells. After 35 ml of the 1× EGTA solution was passed through the liver, it was replaced with 7–10 ml of 1× Leffert’s buffer to flush out the EGTA, which otherwise chelates the calcium ions necessary for collagenase activity in the next step when the liver is digested by perfusion with 25–50 ml of Liberase solution (∼4.3 Wünsch units). After perfusion, the liver was removed from the abdominal cavity and passed through Nitex 80-μm nylon mesh, using extra ice-cold Leffert’s buffer with 0.02% CaCl2and a rubber policeman. Hepatocytes were purified from the remaining cells by two consecutive centrifugations for 4 min, 50 × <em>g</em> each, leaving the other, smaller cell types in suspension. After each spin, the solution was decanted as waste, and the enriched cell pellet of hepatocytes was resuspended in 30 ml ice-cold Leffert’s buffer with 0.02% CaCl2. After the second centrifugation, the cell pellet contained >98.6% hepatocytes.</p>
<p id="__p14">For cardiomyocyte isolation 8-week-old female mice were euthanized and the chest opened to expose the heart. The descending aorta and inferior vena cava were cut and an EDTA buffer was injected into the apex of the right ventricle to flush the heart. The ascending aorta was clamped and the heart transferred to a petri dish and fixed by perfusion of EDTA buffer containing 4% formaldehyde via the left ventricle. The formaldehyde was quenched by perfusing the heart with 125 mM glycine, and digested by perfusion with collagenase buffer. The ventricles were rent into smaller pieces, and triturated to complete cellular dissociation into a single-cell suspension. Cells were then filtered through a 100-μm strainer to remove tissue fragments and centrifuged at a very low speed to obtain a highly enriched fraction of fixed cardiomyocytes.</p>
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