title: GSM8096034: aortic smooth muscle cells library2, HTO; Mus musculus; OTHER
library_name: GSM8096034
library_construction_protocol: B6.Ldlr-/- SMMHC CreERT2Rosafl-mT-stop-fl-mG mice were each i.p. injected with 1 mg Tamoxifen (solved in peanut oil) per day for 5 consecutive days. In this reporter strain, the red fluorescent protein tomato is ubiquitously expressed at baseline and switches to enhanced green fluorescent protein (GFP+) in all cells that have undergone estrogen receptor-dependent Cre-mediated recombination as well as their progeny. 5 days after the last injection, mice were fed a HFD for 3 months and treated with an irrelevant isotype control antibody (250 µg rat IgG2b anti-Phyt1, clone AFRC Mac51, i.p. once weekly) or an CD8+ T cell depleting antibody (250 µg anti-CD8 antibody , clone YTS156.7.7, i.p. once weekly) for 1 month before sacrifice. After euthanasia, arteries were flushed with PBS, aortas were harvested and aortas were separately processed. Aortas were minced and digested for 45 min at 37°C in RPMI-1640 containing 4 U/ml liberase, 60 U/ml hyaluronidase and 120 U/ml DNase I10. Digested aortas were passed through a 70 µm cell strainer to obtain single cell suspension. Each sample was labeled with a specific hashtag antibody (Hashtag #8-11: isotype treated control, Hashtag 12-15: CD8+ T cell depleted, final concentration 1:50, Biolegend) by incubation for 25 min at 4°C. After washing the cells twice, control and CD8+ T cell depleted samples were each pooled and stained with Dapi (dilution 1:1000). Total viable GFP+ cells were sorted in PBS containing 0.04 % BSA using FACS Aria III (BD Bioscience) with a 100 µm nozzle. For each group 15,000 cells were loaded into the 10x Genomics Chromium at a concentration of 1000 cells/µl and cell sequencing was performed as described above. 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. -HASH-seq library: amplification was performed at 95°C for 3' followed by ten cycles 95°C for 20''; 64°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC-3' TruSeq D701_s primer as i7 primer (Illumina). The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3' SI-PCR primer as i5 primer (Illumina) was used for HASH- seq library amplification. Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% HTO library before sequencing.