Plaques were dissected from aortas of aged (> 78 weeks) ApoE-KO mice (n = 3). Plaques were removed using a dissection microscope and cut into small pieces, then digested with enzyme cocktail (400 U/mL collagenase type I, 10 U/mL collagenase type XI, 60 U/mL hyaluronidase and 60 U/mL DNase I, 20 mM HEPES in DPBS) for 40 min at 37°C with slow shaking. Single cell suspensions were stained with fixable viability dye and the panleukocyte marker antibody CD45 (Ebioscience). Total single live CD45+ cells were sorted by FACSAriaTM II. The RNA library of total CD45-positive (CD45+) live cells was constructed using the Chromium Single Cell 5' Library & Gel Bead Kit (10X Genomics) and sequencing was performed by 26+ 91 bp paired-end next generation sequencing (Illumina). The sequencing raw data was demultiplexed with the cellranger mkfastq pipeline, reads were aligned to mouse mm10 reference transcriptome using STAR alignment built in Cell Ranger (version 3.1.0). All 578 macrophages from total plaque CD45+ cells were sub-clustered for 2000 highly expressed genes of each cell using non-linear dimensional reduction (tSNE) algorithm. Three macrophage subtypes were designated following a previous report using marker gene profiles 31. LGALS3/Gal3 gene expression of each macrophage in murine plaques was shown as violent plots.

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