title: GSM5543540: F7_2; Mus musculus; RNA-Seq
library_construction_protocol: Aortas from Ldlr-/- mice fed with a high fat, high cholesterol diet (Altromin, 15% milk fat, 1.25% cholesterol) for 13 weeks were collected, 2 aortas were pooled to generate each sample, and n=5 pools were analyzed. To exclude circulating leukocytes from analysis, mice were injected with 2.5µg anti-CD45.2 APC (clone 104, BioLegend) intravenously 5 minutes before sacrifice to label blood borne immune cells. Aortas were enzymatically digested in RPMI medium containing 450U/ml collagenase I (Sigma-Aldrich C0130), 125U/ml collagenase XI (Sigma-Aldrich C7657), 60U/ml Hyaluronidase (Sigma-Aldrich H3506) for 1 hour at 37°C with agitation, washed in PBS/1% FCS and passed through a 70µm cell strainer. Cells were labeled for 20 minutes at 37°C with CalceinViolet-AM (BioLegend 425203) according to the manufacturer's instructions. Cells were washed and resuspended in PBS 1% FCS, plated in round bottom 96 well plates, and incubated for 10 minutes on ice with anti-CD16/32 (Biolegend, Clone 93, 10µg/ml) to block unspecific binding of antibodies to Fc receptor. Cells were subsequently labeled in a final volume of 100µl for 15 minutes at 4°C with anti-CD45.2 Alexa488 (2.5µg/ml, clone 104, BioLegend), anti-Ter119 PE-Cy7 (1µg/ml, clone TER119, BioLegend), 1:1000 Fixable Viability Dye, CITE-seq antibodies (see table), and hashtag antibodies. Each sample was labeled with a specific Hashtag antibody (Hashtag 1 to 5, final concentration 1:200, BioLegend). Cells were washed twice and all the sample were pooled. Viable Ter119negCD45.2-APC(i.v.)negCalceinAM+ cells were sorted using a BD FACS Aria III with a 100µm nozzle. The amount of sorted cells was adjusted so that ~50% of the total cells represent leukocytes (CD45-Alexa488+). Cells were sorted in PBS supplemented with 0.04% BSA, washed and counted in Trypan blue to assess viability (>85%). 18,000 cells were loaded at a concentration of 600 cells/µl in the 10x Genomics Chromium (Single Cell 3' v3 reagents, 10x Genomics) and scRNA-seq ADT and HTO libraries were prepared. Single cells were encapsulated into droplets with the Chromium™ Controller (10x Genomics). Transcripts captured in all cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 12 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer.