scRNA-Seq of mouse immortalized aortic smooth muscle cells treated with IL1b

<strong>Methods</strong> Mouse immortalized aortic smooth muscle cells (MOVAS; ATCC cell line CRL-2797) were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 200 μg/ml geneticin in a 37 °C incubator with 5% CO2. Cells were cultured in 12-well plates to approximately 70% confluency. Prior to experimental treatments, cells were incubated for 24 h in serum-starvation medium (DMEM supplemented with 0.2% BSA). Subsequently, the growth medium was replaced with serum-starvation medium supplemented with recombinant interleukin-1β (IL-1β; Sino Biological #10139HNAE) at 25 or 50 ng/ml and incubated for a further 24 or 48 h. The different IL-1β treatments were timed to end concurrently and, after trypsinization, the cells were pooled in equal counts to obtain a mixture of cells at different phases of the IL-1β response. As a control treatment, 24 h serum-starved cells were placed in fresh serum starvation medium for a further 48 h. The Chromium Single Cell 3’ Kit (v3 Chemistry; 10x Genomics) was used to prepare scRNA-Seq libraries for control and IL-1β treated cells in separate lanes. Libraries were sequenced on an Illumina NextSeq 550 instrument and reads were processed using the Cell Ranger pipeline (version 3.0.2; 10x Genomics) and the mm10 reference package (version 3.0.0). Additional cell quality filtering was carried out using Seurat (version 3.1.0; PMID 31178118), retaining the cell barcodes with 1000-5000 genes, 2000-12500 UMI-s and &lt;15% mitochondrial reads. <strong>File format</strong> Tab-separated non-sparse matrices of raw counts for scRNA-Seq cells that passed cell QC (described above). Files are compressed with gzip. <strong>Samples</strong> 1) MOVAS_IL1b_24h_48h_pooled [1355 cells (columns) and 31053 genes (rows)] 2) MOVAS_serum_starved_control [1337 cells (columns) and 31053 genes (rows)] <strong>Publication reference</strong> To be added.