title: GSM9405354: Mouse aorta, Tcells, vdj, control; Mus musculus; OTHER
library_construction_protocol: In Vivo BiTE Treatment Studies Animal studies were performed in compliance with guidelines set forth by the National Institutes of Health Office of Laboratory Animal Welfare and approved by the Washington University institutional animal care and use committee. Animals were housed in a controlled environment with a 12 h light–dark cycle, with free access to water and a standard chow diet.  All experimental mice were fed a high fat diet containing 21% fat by weight (42% kcal from fat) and 0.2% cholesterol (#TD.88137, Envigo Teklad) for 4, 16, or 20 weeks (For Apoe-/-) starting at 8 or 9 weeks of age. For all studies an isotype BiTE was used for the control group and an anti-FAP BiTE for the treatment group. Animals were injected intraperitoneally and dosed at 400 μg/kg. For the acute model animals were treated with BiTE once a week starting week 1 for a total of 4 injections. For chronic studies mice were treated once a week in the same manner starting at 12 weeks of HFD feeding.  Single cell RNA-seq in murine atherosclerosis The ascending aorta (from beginning of aorta up to aortic arch including first intercostal artery) were isolated and transferred to an enzymatic dissociation cocktail with 250 U ml-1 Collagenase IV (#C5138, Sigma Aldrich), 60 U ml-1 Hyaluronidase (#H3506), and 60 U ml-1 DNase I (#D4527, all purchased from Sigma Aldrich) in Dulbecco's Modified Eagle Medium (DMEM) and slightly minced. After incubation at 37 °C for 60 min with agitation, the digestion reaction was quenched with 6 mL of HBB buffer (2% Fetal Bovine Serum (FBS) and 0.2% Bovine Serum Albumin (BSA) in Hank's Balanced Salt Solution (HBSS)), then filtered through 70 μm filters. Samples were pelleted by centrifugation at 4 °C, 1,400 rpm for 5 min and the supernatant was discarded. Cells were resuspended in FACS buffer (2% FBS and 2mM Ethylenediaminetetraacetic acid (EDTA) in calcium/magnesium free PBS) and centrifugation was repeated in above conditions and supernatant aspirated. Cell pellet was resuspended in 100 µL FACS buffer with the addition of 1 µL of DRAQ5 (#564907, Thermo Fisher Scientific) and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and 1 µL DAPI (#564907, BD Biosciences,) and filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. Collected cells were processed using the single Cell 3' Kit v 3.1 (#1000268, 10x Genomics). 10,000 cells were loaded onto ChipG (#1000121) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA was performed as recommended in the 3' v3.1 chromium protocol. Libraries were sequenced on a NovaSeq 6000.  Isolation of T-cells for scRNA-seq and TCR-seq in murine atherosclerosis The ascending aorta (from beginning of aorta up to aortic arch including first intercostal artery) were isolated and transferred to an enzymatic dissociation cocktail with 2 U ml-1 Liberase (#5401127001, Sigma Aldrich), and 2 U ml-1 Elastase (#LS002279, Worthington) in DMEM and slightly minced. After incubation at 37 °C for 45 min with agitation, the digestion reaction was quenched with 6 mL of HBB buffer, then filtered through 70 μm filters. Samples were pelleted by centrifugation at 4 °C, 1,400 rpm for 5 min and the supernatant was discarded. Cells were resuspended in FACS buffer and centrifugation was repeated in above conditions and supernatant aspirated. Cell pellet was resuspended in 100 µL FACS buffer with the addition of 1 µL of CD45, CD11b, and CD3 and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and filtered into filter-top FACS tubes. First singlets were gated and subsequent CD45+/CD11b-/CD3+ events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. Collected cells were processed using the single Cell 5' Kit v 2 (#1000244, 10x Genomics). 10,000 cells were loaded onto ChipK (#2000182) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA were performed as recommended in the 5' v2 chromium protocol. V(D)J amplification and library construction was performed per protocol (#1000252, 10x Genomics). Libraries were sequenced on a NovaSeq 6000. For scRNA-seq: Collected cells were processed using the single Cell 3' Kit v 3.1 (#1000268, 10x Genomics). 10,000 cells were loaded onto ChipG (#1000121) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA was performed as recommended in the 3' v3.1 chromium protocol. Libraries were sequenced on a NovaSeq 6000.  For TCR: Collected cells were processed using the single Cell 5' Kit v 2 (#1000244, 10x Genomics). 10,000 cells were loaded onto ChipK (#2000182) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA were performed as recommended in the 5' v2 chromium protocol. V(D)J amplification and library construction was performed per protocol (#1000252, 10x Genomics). Libraries were sequenced on a NovaSeq 6000.