GSM7898091: CD146_HIC1_YFP_undamaged; Mus musculus; RNA-Seq
Mice were anesthetized with 0.5 mg/g tribromoethanol (Avertin) IP.  Following transcardiac perfusion with 20 mL of PBS-EDTA (2mM), whole hearts were excised and their atria were trimmed off. For single-cell RNAseq (scRNAseq), excised ventricles were cut into 2 mm pieces and digested in Collagenase type II solution (Millipore Sigma C6885; 500uL per heart at 2.5 U/mL) containing 5 mM CaCl2 for 30 minutes at 37°C. Digested lysate was quenched and centrifuged at 140-g to remove the supernatant. This was followed by a second round of digestion in a solution (500 uL per heart) containing Collagenase D (Millipore Sigma 11088882001; 1.5 U/ml), Dispase II (Millipore Sigma 04942078001; 2.4 U/ml) and 5 mM CaCl2 for 1 hour at 37°C. Digested lysate was quenched and filtered through 40-μm strainer filters. Filtrate was centrifuged at 500-g and washed again with cold FACS buffer prior to incubation with primary antibodies for 30 minutes. Stained cells are double-sorted using a BD Influx (Becton Dickinson) into DMEM (Gibco 11965-092) containing 20% fetal bovine serum (FBS; Sigma F1051). Cell-hashing with oligo-tagged antibodies against MHC class I and CD45 (BioLegend 155801/155803/155805/155807) were applied in some samples. For scRNAseq, viable cells were delivered into a Chromium Controller (10X Genomics), captured and libraried with the Chromium single cell 3' reagent kit v2 or v3 (10X Genomics) as per the manufacturer's protocol. Sample integrity was assessed on the Agilent Bioanalyzer 2100 system using the RNA 6000 Pico or Nano kit (Agilent, 5067-1511 and 5067-1513, respectively). cDNA libraries were sequenced on Illumina Nextseq 500 using the High Output 75 Cycle kits to a minimum depth of 30,000 mean reads per cell.