Experimental work was carried out by H.F. Jørgensen, A.L. Taylor, J.L. Harman and J. Chappell, unless otherwise stated. In case of the carotid arteries and the whole aorta 10X Genomics Chromium experiments, the entire artery was dissociated to a single-cell suspension by incubation with collagenase type IV (2.5 mg/ml) and porcine pancreatic elastase (2.5 U/ml) for 1-2h, followed by filtering through a 40 µm cell strainer to remove any remaining clumps of cells. In other experiments, the adventitial and endothelial layers were removed prior to dissociation of medial cells to a single-cell suspension as described above.
For SCA1 antibody staining, single-cells were incubated with TruStain FcX (1:100, BioLegend) prior to staining with the APC-conjugated isotype control antibody (1:10 Miltenyi 130-102- 655) or the anti-SCA1 antibody (1:10 Miltenyi 130-120-343). SCA1 antibody staining was analysed using an Accuri C6 (wild type and the single-colour reporter) or a Fortessa (multi- colour reporter) flow cytometer.
2.3.1 Fluidigm C1 platform
Medial layers of the AA and DT aortic sections from 5-7 male C57Bl/6 mice were isolated and dissociated to a single-cell suspension, as described above. Samples (100 cells/µl) were processed using the Fluidigm C1 Auto Prep Arrays system with medium-sized chips (17-25 µm) in line with manufacturer’s instructions. Visual inspection of the chips was performed to select successfully captured single-cells. SMARTer Ultra Low RNA Kit (Clontech) was used to generate amplified cDNA. Sequencing libraries were then prepared using the Nextera Library Prep Kit (Illumina) and sequenced on a HiSeq 2500 sequencer (Illumina) using the 50bp paired-end sequencing protocol. Single cells from both the AA and DT regions were analysed in two independent experiments.
For the pooled VSMC and adventitial samples, single-cell suspension was prepared from the medial and adventitial layers of the aorta, as described above. Total RNA from 2000-4000 cells was then extracted with the RNeasy Plus Micro Kit (Qiagen). Amplified cDNA was generated using the SMARTer Ultra Low RNA Kit (Clontech) according to the tube control protocol. Nextera Library Prep Kit (Illumina) was used to prepare sequencing libraries from the amplified
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cDNA. Libraries were then sequenced on a HiSeq 2500 sequencer (Illumina) using the 50bp paired-end sequencing protocol.
2.3.2 Smart-seq2 platform
Medial layers of 5-7 Myh11-CreERt2/Confetti male mice were dissociated to a single-cell suspension and stained for SCA1 as described above. Cells were additionally stained with Zombie-NIR (1:100, BioLegend) to identify dead cells. Aria-Fusion flow cytometer (BD Bioscience) was used to sort live single cells, which were lineage-labelled and/or SCA1- positive, into individual wells of a 96-well plate. Lineage-labelled cells were required to express only one of the four confetti colours to reduce the chances of profiling doublets of cells. Amplified cDNA was generated using the Smart-seq2 protocol (Picelli et al. 2014), with these modifications: for the reverse transcription Primescript (Clontech) was used, cDNA was amplified in 24 PCR cycles and ERCC controls (Invitrogen) were added at 1:40,000,000 or 1:80,000,000 dilution into the reverse transcription (RT) mix. Sequencing libraries were prepared with the Nextera Library Prep Kit (Illumina) and sequenced on a HiSeq 2500 sequencer (Illumina) through the 50bp paired-end sequencing. Analysed cells were from 3 independent experiments, with SCA1-positive, lineage-labelled cells included in all three experiments and cells, which were only positive for either SCA1 or the lineage label, included in two of the experiments.
2.3.3 10X Genomics Chromium platform
The 10X Genomics Chromium platform with the Gene Expression v2 kit was used to generate the datasets described in this section.
2.3.3.1 VSMCs in healthy aorta and whole aorta analysis
For both the whole aorta and VSMC-only samples, aortas of three tamoxifen-labelled Myh11- CreERt2/Confetti males were processed to a single-cell suspension as described above and stained with Zombie-NIR (1:100, BioLegend). Aria-Fusion flow cytometer (BD Bioscience) was used to isolate 20,000 live single cells for both samples. In case of the whole aorta sample, selection based on lineage label expression was not performed, while expression of one of the four fluorescent proteins was required for the VSMC-only sample to reduce the chances of profiling doublets. Samples were then processed through the 10X Genomics Chromium
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platform. Sequencing libraries were sequenced on a HiSeq4000 instrument (Illumina) using paired-end sequencing.
2.3.3.2 Carotid ligation injury
The carotid ligation surgery has been performed on the left carotid artery as described previously and above (Kumar & Lindner 1997; Chappell et al. 2016). Left carotid arteries from 5 male tamoxifen-labelled Myh11-CreERt2/eYFP/Ki67-RFP mice were dissected 7 days following carotid ligation and dissociated to a single-cell suspension as described above. An Aria-Fusion flow cytometer (BD Bioscience) was used to isolate 20,000 live single eYFP+ cells. Due to the rare nature of RFP+ cells, the sample was enriched for about 100 eYFP+RFP+ cells. Isolated cells were processed through the 10X Genomics Chromium system and their libraries were sequenced on a HiSeq4000 sequencer (Illumina) using paired-end sequencing.
2.3.3.3 VSMCs in atherosclerotic arteries
Myh11-CreERt2/Confetti/ApoE-/- tamoxifen-labelled male mice were fed a high fat diet for
either 14 or 18 weeks as described above. Aortas of 2-3 mice per time point were used, with atherosclerotic plaques manually isolated, dissociated essentially as described previously (Butcher et al. 2011), with the addition of porcine pancreatic elastase (2.5 U/ml) to the digestion cocktail, and passed through a 40 µm cell strainer to remove clumps. 20,000 single lineage-labelled cells, which expressed only one of the four fluorescent proteins, were isolated from each time point using an Aria-Fusion flow cytometer (BD Bioscience). Samples were processed through the 10X Genomics Chromium platform and sequenced using pair-end sequencing on a HiSeq4000 machine (Illumina). Experiment performed jointly with H.F. Jørgensen.