title: GSM8966235: non-cardiomyocytes from digested mouse heart harvested 20 days after sham surgery; Mus musculus; RNA-Seq
library_construction_protocol: adult non-cardiomyocytes were isolated using the Langendorff-free method61, with some modifications: After euthanizing mice, the chest was opened to expose and cut the descending aorta. The ascending aorta was clamped with a Reynolds forceps and the right ventricle was injected with 15 ml of EDTA buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4 , 10 mM HEPES, 10 mM Glucose, 10 mM BDM, 10 mM taurine, 5 mM EDTA). The perfused heart was then transferred to a plastic dish containing EDTA buffer and 10 mL perfusion buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 10 mM HEPES, 10 mM glucose, 10 mM BDM, 10 mM taurine, 1 mM MgCl2) were injected into the left ventricle. This was followed by repeated (approximately 4) injections of 10 ml digestion buffer (perfusion buffer plus collagenase II 0.5 mg/mL (Worthington Biochemical Corporation, Cat. LS004177), collagenase IV 0.5 mg/Ml (Sigma Aldrich, C5138), protease XIV 0.05 mg/mL) (Sigma Aldrich, P5147)) until the heart became pale and soft. The ventricle was then minced with a forceps and cellular dissociation was completed by gentle trituration of heart fragments. 5 mL stopping buffer (perfusion buffer with 5% FCS) were added to terminate digestion. The cell suspension was passed through a 100-μm filter and cardiomyocytes were allowed to settle by gravity for 20-30 minutes at room temperature. Non-cardiomyocytes were collected from the supernatant, followed by red blood cells lysis using RBC lysis buffer (Thermo Scientific Chemicals, J62150.AP) and debris removal using Debris Removal Solution (Miltenyi, 130-109-398). Dead cells were stained with DAPI and viable single cells were then sorted by a flow cytometer (BD, FACSAria III). The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer's protocol to obtain 10.000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics). Single cell RNAseq library preparation was done using a standard protocol.