biostudies-arrayexpressDiana SharyshMus musculusSeurat 4.2.2Cell Ranger 6.1.1https://www.ebi.ac.uk/biostudies/studies/E-MTAB-17142Smooth muscle cells (SMCs) undergo phenotypic switching during atherosclerosis and give rise to a large fraction of plaque cells. To investigate the role of hypoxia-inducible factor 1α (HIF1α) signaling in SMC behaviour during atherogenesis, we used mice in which Hif1a is conditionally deleted in lineage-traced SMCs (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed). Cre recombination was induced with tamoxifen at 6 weeks of age. Atherosclerosis was induced by a single tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet feeding. Aortic arches were pooled by genotype from SMC-specific Hif1a knockout (Hif1aSMC-KO, n=8) and wild-type control (Hif1aWT, n=6) male mice, enzymatically dissociated, and viable cells (Draq5+/DAPI–) isolated by fluorescence-activated cell sorting. Single-cell transcriptomes were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform and sequenced on an Illumina HiSeq 4000. Reads were aligned to the mm10 reference transcriptome supplemented with the tdTomato transgene sequence using Cell Ranger. The dataset enables comparison of cell-type composition and of SMC-derived cell transcriptional states between genotypes, with a focus on hypoxia- and stress-response pathways underlying SMC phenotypic modulation. Only male mice were studied because the Myh11-CreERT2 transgene is Y-linked.biostudies-arrayexpressSample Treatment - Cre recombination was induced by intraperitoneal injection of tamoxifen (2 mg in corn oil) on five consecutive days from 6 weeks of age. To induce atherosclerosis, mice received a tail-vein injection of rAAV8-PCSK9 (1×10¹¹ vector genomes) at 9 weeks of age followed by high-fat diet (SSNIFF S9167-E011) for 21 weeks. Aortic arches were harvested at 30 weeks of age for single-cell analysis.Nucleic Acid Extraction - Up to approximately 10,000 sorted viable cells per sample were loaded onto a Chromium Next GEM Chip G and partitioned into Gel Beads-in-Emulsion using the 10x Genomics Chromium controller. Cells were lysed within GEMs and mRNA was captured and reverse-transcribed into barcoded cDNA using the Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics) according to the manufacturer's instructions.Library Construction - Following cDNA amplification, 3′ gene expression libraries were constructed using the Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics) according to the manufacturer's instructions. Library size distribution was assessed on a 2100 Bioanalyzer (Agilent) and library concentration was quantified using a Qubit fluorometer (Thermo Fisher Scientific).Growth Protocol - Mice were group-housed under specific pathogen-free conditions with a 12-h light–dark cycle at 23 ± 1°C and 50 ± 5% humidity, with ad libitum access to water and food (SAFE D184 when not on high-fat diet) at CNIC. Only male offspring were studied, as the Myh11-CreERT2 transgene is inserted on the Y chromosome.Sample Collection - Aortic arches were harvested from male mice with smooth muscle cell–specific deletion of Hif1a (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed; Hif1aSMC-KO) and Cre-positive wild-type controls. Atherosclerosis was induced by tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet. Arches were pooled by genotype (Hif1aWT n=6, Hif1aSMC-KO n=8), cleaned in situ of fat and surrounding tissue, and pretreated with 2 mg/ml collagenase II in HBSS for 15 min at 37°C to remove the adventitia. Stripped arches were washed, opened longitudinally, cut into small pieces, and digested in HBSS containing 200 µg/ml hyaluronidase, 250 µg/ml Liberase, and 200 µg/ml DNase for 60 min at 37°C. The suspension was filtered through a 70 µm strainer, stained with DAPI (1 µg/ml) and Draq5 (5 µM), and viable cells (Draq5+/DAPI–) were isolated on a FACSAria cell sorter. Viability (>80%) was confirmed on a Countess III counter.Sequencing - Libraries were sequenced in paired-end mode on an Illumina HiSeq 4000. Base calling was performed with RTA v1.18.66.3, and per-sample FASTQ files were generated using the Cell Ranger mkfastq pipeline (10x Genomics).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Cell Ranger filtered barcode count matrices were processed in Seurat (v4.2.2). Cells were filtered on quality metrics (1200 < UMIs < 25000; 1000 < genes < 5000; <15% mitochondrial; <0.1% haemoglobin; library complexity > 0.8; mito/ribo ratio < 0.75; <10% Malat1) and doublets removed by consensus of DoubletFinder and scDblFinder (3525 KO, 2802 WT cells retained). Counts were normalized and scaled, and samples integrated using the Seurat RPCA algorithm (k.anchor = 5), followed by dimensionality reduction and clustering.Sequence Alignment - Raw FASTQ files were processed with Cell Ranger (v6.1.1, 10x Genomics) against the mm10 reference transcriptome (2020) supplemented with the tdTomato transgene sequence.UnknownTranscriptomicsGenomicsProteomicsBD FACSAria cell sorter (viable-cell sorting); Countess III cell counter (Thermo Fisher; viability check)Illumina HiSeq 400010x Genomics Chromium controller; Agilent 2100 Bioanalyzer and Qubit fluorometer (Thermo Fisher) for QCNaN10x Genomics Chromium controller (GEM generation on Chromium Next GEM Chip G)RNA-seq of coding RNA from single cellsMus musculusJacob Fog BentzonDiana SharyshfalsescRNA-seq of aortic arches from smooth muscle cell–specific Hif1a knockout and control mice with atherosclerosisSmooth muscle cells (SMCs) undergo phenotypic switching during atherosclerosis and give rise to a large fraction of plaque cells. To investigate the role of hypoxia-inducible factor 1α (HIF1α) signaling in SMC behaviour during atherogenesis, we used mice in which Hif1a is conditionally deleted in lineage-traced SMCs (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed). Cre recombination was induced with tamoxifen at 6 weeks of age. Atherosclerosis was induced by a single tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet feeding. Aortic arches were pooled by genotype from SMC-specific Hif1a knockout (Hif1aSMC-KO, n=8) and wild-type control (Hif1aWT, n=6) male mice, enzymatically dissociated, and viable cells (Draq5+/DAPI–) isolated by fluorescence-activated cell sorting. Single-cell transcriptomes were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform and sequenced on an Illumina HiSeq 4000. Reads were aligned to the mm10 reference transcriptome supplemented with the tdTomato transgene sequence using Cell Ranger. The dataset enables comparison of cell-type composition and of SMC-derived cell transcriptional states between genotypes, with a focus on hypoxia- and stress-response pathways underlying SMC phenotypic modulation. Only male mice were studied because the Myh11-CreERT2 transgene is Y-linked.2026-06-21T00:00:00Z2026-06-21T01:00:52.424Z2026-06-10T14:58:33.632ZE-MTAB-17142ERP194840EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969