Cardiac Nuclei Isolation and scRNA-seq

Cardiac tissues were excised from Cdh5-Cre^ERT2;Pdgfrb^fl/fl and Pdgfrb^fl/fl mice, cut into small pieces, and homogenized using a tissue grinder (Sigma, D8938) in isolation buffer (0.32 M sucrose, 5 mM CaCl₂, 3 mM MgAC₂, 0.1 mM EDTA, 0.1% Triton X-100, and 10 mM Tris-HCl pH 8.0) supplemented with cOmplete protease inhibitor cocktail (Sigma, 11697498001). Tissues were processed with homogenizers pestle A for 15 strokes and pestle B for 25 strokes to release the nuclei, followed by filtration with a 40-μm Flowmi cell strainer (SPBel-Art, H13680–0040), centrifugation at 500 × g for 5 min at 4 °C, and washing with 0.01% BSA in PBS. The nuclei were counted and diluted to 700–1,200 nuclei/μl and analyzed followed by a 3’ GEX library protocol (10x Genomics, V3.1) for single-nuclei RNA-seq at the next-generation sequencing core at the Children’s Hospital of Philadelphia. Data were analyzed using CellRanger software 6.1.2 (10x Genomics) and a pipeline that produces sparse numerical matrices for each sample, with gene-level counts of unique molecular identifiers (UMI) identified for all single cells passing default quality control metrics. These gene expression matrices were processed with Seurat 4.0 and Monocle3 R package ^{54 (link)}.