title: Mouse, pig, and human atherosclerotic lesions have common and distinct mesenchymal cell populations
design: Mouse, pig, and human atherosclerotic lesions have common and distinct mesenchymal cell populations
library_name: Sample_Pig7_RCA_p
library_construction_protocol: Animals were euthanized, and the right coronary arteries were quickly isolated and opened. The atherosclerotic plaques were dissected without media. Approximately 200-250 mg of plaque was selected, washed in HBSS, and minced. It was incubated for 60 minutes at 37 °C in 1 ml of an enzyme solution containing 1,5 mg/ml (8 U/ml) of elastase (LS002279, Worthington Biochemical), 2 mg/ml of liberase (05401119001, Sigma Aldrich) and 300 µg/ml of DNaseI (Sigma Aldrich, DN25). Non-digested tissue was disaggregated using a smooth-tipped Pasteur pipette. Reactions were stopped by adding PBS containing 0.5% bovine serum albumin (BSA). The suspension was passed through a 70 µm pore filter to remove aggregates. Cells were collected by centrifugation and resuspended in 500 µl PBS+0.5% BSA. DAPI (final concentration, 1 µg/ml final ) and Draq5 (Thermo Scientific 65-0880-96; final concentration, 5 µM) were added to enable detection of viable and dead cells. Viable cells (Draq5 positive and DAPI negative) were sorted with a FACSAria Cell Sorter. Atherosclerosis was induced in PCSK9D374Y transgenic minipigs by feeding a standard pig feed supplemented with 20% w/w lard, 1.5% w/w cholesterol (HFHC), 0.7% NaCl w/w, from 3 until 18 months of age. Up to 16500 cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics). After reverse transcription and cDNA amplification, sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).