Single-cell RNA-seq of aortic CD3+ T cells
Single-cell RNA-seq of aortic CD3+ T cells
Chimeric Ldr-/- CD45.1 mice were grafted with BM from Xcr1Cre-DTA or control Xcr1Cre mice. After BM reconstitution, these chimeric mice were fed HCD for 8 weeks and aortae were collected. The aortas were kept in cold Dulbecco's Modified Eagle Medium (DMEM, Gibco) before being cut into smaller pieces that were incubated in digestion buffer (Collagenase A (25 mg/ml; Sigma-Aldrich), Dispase II (25 mg/ml; Sigma-Aldrich), DNase I (250 μg/ml), elastase (25 μg/ml), and Liberase TL (0.2 units/mL; Wünsch) for 30 min at 37ºC in a water bath. The digested tissue was mixed by pipetting, filtered through a 70 μm strainer and centrifuged at 400g for 5 min at 4ºC.To achieve sufficient cell numbers, 10 aortae per genotype were pooled and CD3+ T cells were sorted. Briefly, the pool of aortae was pre-purified using MACS® Cell Separation CD45 MicroBeads (Miltenyi Biotec). The CD45+ fraction was collected, and cells were stained in FACS Buffer with anti-CD16/32 and antibody cocktail containing CD45-BV421 (Biolegend #103133), CD11c-FITC (Biolegend #117305), NK1.1-FITC (Biolegend #108705), I-A/I-E-FITC, Ly6C-FITC, Ly6G-FITC, F480-FITC, B220-FITC (Biolegend #103205), CD3-PECy7 (Biolegend #100219) and DAPI as a live/dead marker. CD45+ CD3+ CD11c- I-A/I-E–F480-Ly6C-Ly6G-NK1.1- cells were sorted using FACSAria™ Fusion Cell Sorter (Becton Dickinson). Cells were counted, and their viability was checked using the Countess III cell counter (Thermofisher). Each cell suspension was loaded into one port of a Chromium Next GEM Chip G (10x Genomics). Single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). Sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) following the manufacturer instructions. The average size of each library was then calculated using a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and the concentration was determined using the Qubit fluorometer (Thermofisher)