title: Single-cell RNA sequencing of Versican (Vcan) knockout mice after cardiac ischemia.
design: Single-cell RNA sequencing of Versican (Vcan) knockout mice after cardiac ischemia.
library_name: Rot4KO_p
library_construction_protocol: Cardiac interstitial cells were isolated from WT and Vcan-KO mice (n=2,3) 7 days after cardiac ischemia with reperfusion. 10 min prior to harvest, mice were injected with 250 IU heparin i.p., hearts were perfused with cold PBS for 2 min before removal to a glass dissection plate. Atria and valves were discarded and ventricular tissue was moved to a fresh 100 mm petri dish on ice with a cold droplet of PBS. Ventricles were then minced until 1 mm cubes and then moved to a pre-chilled 2 mL tube containing a digestion solution, consisting of 600 U/mL collagenase type I (Worthington) and 60 U/mL DNase I (Sigma) in HBSS with calcium and magnesium. Tissue was then digested at 37 C for 20 min while rocking, with vigorous pipetting to disrupt the tissue every 10 min. The cell suspension was then filtered over a 100 µm filter sitting in a 50 mL falcon tube, the volume was then adjusted to 15 mL with cold PBS before filtering again with a 40 µm filter. Cells were then pelleted at 800 g for 10 min at 4 C. Supernatant was discarded, and erythrocytes were lysed for 7 min at 4 C. Cells were pelleted again at 300 g for 10 min at 4 C, resuspended in PEB, and incubated with 7-Aminoactinomycin D (Invitrogen), to excluded dead cells and anti-CD31 and anti-CD45 antibodies. Single, live, CD31-, CD45- cells were isolated by FACS for input into the chromium controller. Performed using Chromium Single Cell 3' NextGEM Reagent Kit v3 according to the manufacturer's instructions. Performed using Chromium Single Cell 3' NextGEM Reagent Kit v3 according to the manufacturer's instructions.