GSM5609417: non-CMs, Ctrl-MI; Mus musculus; RNA-Seq
The isolation of non-CMs from the murine heart was performed as previously described with some modifications (Forte et al., 2020a; Skelly et al., 2018). In brief, mice were sacrificed by cervical dislocation, and hearts were immediately cannulated and perfused with cold PBS (50 ml). Remove the heart, separate the atria and valves from the heart, and cut the ventricular myocardium into 1 mm pieces on a bacterial Petri dish on ice. Transfer to a 10 ml tube containing 3 ml of enzyme solution and incubate in a water bath at 37°C for 45 min, trituration every 15 min. Enzyme solution contained 2 mg/ml collagenase type IV (Worthington Biochemical, CLS-4) and 1.2 U/ml Dispase II (Sigma, 255-914-4). After the final trituration, the enzyme/tissue mixture is filtered through a 40 μm cell strainer, FACS buffer and dead cell removal solution (Miltenyi Biotec, 130-109-398) is added, and debris is removed after centrifugation. Further, use RBC lysis buffer (pluriSelect, 60-00050-11) to remove red blood cells, then dye the cell suspension with Live/Dead (Invitrogen, L34975,1:1000), Calcein Violet Working solution (BioLegend, 425203, 0.1μM). Using a BD AriaIIu with a 130 μm nozzle, cells with intact cell membranes and metabolically active cells (Live/Dead-, Calcein+) were sorted (12 psi) while minimizing pressure on the cells. Libraries from non-CMs suspensions were generated with a Chromium controller from 10X Genomics according to the manufacturer's instructions. Approximately 8,000 whole cells were loaded into each channel and processed using the Chromium Single Cell 3' v3.1 reagent kit (10X Genomics). single cell RNA-seq