GSM8453030: human_ST_Section_C_10X; Homo sapiens; OTHER
Sample 1 and 2: Datasets of lineage-labelled VSMCs from healthy and injured mouse carotid arteries were generated as described (Worssam et al. 2023) after pooling cells from 5 Myh11eYFP animals 11 days post-ligation (D11) or 2 control Myh11eYFP animals that did not undergo surgery (NS). After carotid artery harvest and dissection, arteries were incubated for 6 minutes in collagenase type IV (1 mg/mL, Life Technologies) and porcine pancreatic elastase (1 U/mL, Worthington) in DMEM. After removing the adventitia, the medial samples from each animal cohort were pooled and digested in collagenase type IV (2.5 mg/mL, Life Technologies) and porcine pancreatic elastase (2.5 U/mL, Worthington) in DMEM for 90 minutes. Medial cell suspensions were filtered through a 40 µm filter and singlet cells expressing eYFP isolated by fluorescence-activated cell sorting (FACS, Aria-Fusion flow cytometer, BD Bioscience) into PBS with 0.05% BSA and 5 µL/mL recombinant RNAse inhibitor (Sigma). Isolation of single cells was verified with a haemocytometer. Sorted cells (10,000) were pelleted, resuspended in 47 µL of PBS with 0.05% BSA and loaded for Drop-Seq using the 10X Chromium system. Amplified cDNA libraries were sequenced on a HiSeq 4000 system. Sample 3-6: A plaque-containing aortic sample from a 54 year old male organ donor was snap-frozen by fully submerging in an isopentane bath cooled by dry ice until frozen, embedded in OCT (2-8°C), sectioned and analysed using VISIUM technology (cat 1000184) according to manufacturer's instructions (10X). Sample 1 and 2: A total of 10,000 EYFP+ cells were loaded onto a Chromium system (10x Genomics) and amplified cDNA libraries generated using the 3' Gene Expression v3.0 kit (10x Genomics) were sequenced using 150 cycle paired end protocol (NovaSeq, Illumina). Sample 3-6: Frozen sections were processed using the Visium Spatial Gene Expression protocol (10x Genomics), and spatially resolved transcriptomic libraries were constructed and sequenced using a 150-cycle paired-end protocol (NovaSeq, Illumina).