Bone morphogenic protein-4 availability in the cardiac microenvironment controls myocardial inflammation and fibrosis - Single cell transcriptome analysis from healthy and inflamed murine hearts
Bone morphogenic protein-4 availability in the cardiac microenvironment controls myocardial inflammation and fibrosis - Single cell transcriptome analysis from healthy and inflamed murine hearts
Euthanized animals were perfused with 20 ml of PBS before hearts were harvested. For enzymatic dissociation hearts were minced into small pieces and were placed into a six-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM HEPES (Lonza), 1 mg/ml collagenase D (Sigma), and 25 μg/ml DNase I (Applichem) and incubated at 37°C under continuous stirring. The remaining tissue pieces were mechanically disrupted.  For analysis of total cardiac cells, single cell suspensions of freshly isolated murine hearts were sorted for total live cells using a BDFACSMelody. Fibroblast samples were sorted for live CD45-CD31-PDPN+ cells using a BDFACSMelody. Sorted single cells suspensions were emulsified for library generation using the droplet-based 10x Chromium (10x Genomics) system. cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).