Effect of pressure-overload on cardiac endothelial cells in C57BL6/J mice

We performed single-cell RNA-seq analysis of FACS sorted cardiac blood vascular and lymphatic endothelial cells in the heart of adult C57 female mice in a model of pressure-overload. Overall design: Cardiac CD31+ CD45neg endothelial cells, and Lyve1/Podoplanin-expressing lymphatic endothelial cells (LEC) were extracted from hearts of mice 8 weeks after pressure-overload or sham surgery in a murine model of transaortic constriction, and analyzed by single-cell RNA-sequencing. Briefly, cardiac single cell suspensions were sorted by FACS (ARIA II, BD Biosciences). BECs were defined as live CD45-/CD31+/Lyve1- cells, whereas LECs were identified as CD45-/CD31+/Lyve1+/Pdpn+ cells. A 1:3 mix of cardiac LECs and BECs were included in each sample prepared using the 10X Genomics Chromium Next Gem Single Cell 3' kit. For each group (Sham or TAC), 1-2 single cell sorting and GEM reactions were performed, using each time cells pooled from 10 mouse hearts. Samples were sequenced using Illumina NextSeq550 at an estimated sequencing depth of 20 000 reads per cell. Reads were aligned with the mouse genome using STARsolo. For low-quality data filtering, genes expressed by <10 cells, and cells expressing <300 genes, or >10% mitochondria-derived UMI counts, were excluded. R package Seurat was used for read normalization, data integration, and principal component-based unsupervised clustering and statistical tests. Among 4178 total sequenced cells, 1577 cells remained post-filtering, including 665 cells from sham-operated controls and 912 cardiac cells from TAC mice.