GSM8146673: WT_18_days_pregnant_ECs_scRNA-seq; Mus musculus; RNA-Seq
Heart digestion and cardiac endothelial cell isolation was performed as previously described in our study in Circulation (PMID: 33203221 DOI: 10.1161/CIRCULATIONAHA.120.050635). Briefly, hearts from 2-3 mice per condition/genotype were digested and pooled and subsequently treated as 1 sample. The pooled hearts were rinsed with DPBS containing CaCl2, manually minced and digested with a mixture of Collagenases (type I, II, and IV) at 37°C followed by washing and straining. The obtained cells were subjected to downstream steps for SVF or stained and FAC sorted to obtain ECs. The cells were loaded into Chromium Next GEM Single Cell 3' Chip v3.1 (Dual index) (10x Genomics, Pleasanton, CA) and gel beads in emulsion (GEM) generation was performed aiming at 10 000 cell captures/sample. Subsequent cDNA purification, amplification (11 cycles), and library construction (sample index PCR 12 cycles for WT-EC and αMHC-VEGF-B-EC samples, 13 cycles for aP2-VEGF-B-SVF and αMHC-VEGF-B-SVF samples, and 14 cycles for all other samples) was performed as instructed in manufacturer's kit.