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.

<strong>实验方法</strong> 将小鼠永生化主动脉平滑肌细胞（MOVAS；ATCC细胞系CRL-2797）置于添加了10%胎牛血清（Fetal Bovine Serum, FBS）、100 U/ml青霉素、100 μg/ml链霉素及200 μg/ml遗传霉素的达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）中，于37℃、含5%二氧化碳的培养箱内培养。细胞接种于12孔板，培养至约70%汇合度。实验处理前，将细胞置于含0.2%牛血清白蛋白（Bovine Serum Albumin, BSA）的DMEM血清饥饿培养基中孵育24小时。随后将生长培养基替换为添加重组白细胞介素-1β（Interleukin-1β, IL-1β；Sino Biological #10139HNAE）的血清饥饿培养基，IL-1β浓度设为25 ng/ml或50 ng/ml，继续孵育24小时或48小时。所有IL-1β处理组均设置为同步结束处理，经胰酶消化后，按等细胞数混合各组细胞，以获取处于IL-1β应答不同阶段的细胞混合物。对照组处理为：将经24小时血清饥饿的细胞更换为新鲜血清饥饿培养基，继续孵育48小时。采用Chromium单细胞3’试剂盒（v3化学法；10x Genomics）分别为对照组与IL-1β处理组细胞制备单细胞RNA测序（single-cell RNA sequencing, scRNA-Seq）文库，两组文库于不同泳道上样测序。测序在Illumina NextSeq 550仪器上完成，测序reads通过Cell Ranger分析流程（版本3.0.2；10x Genomics）及mm10参考基因组包（版本3.0.0）进行处理。后续采用Seurat分析工具（版本3.1.0；PMID 31178118）进行额外的细胞质量过滤，保留符合以下条件的细胞条形码：检测到1000~5000个基因、2000~12500个UMI、线粒体reads占比<15%。
<strong>文件格式</strong> 通过上述细胞质量控制的scRNA-Seq细胞原始计数采用制表符分隔的非稀疏矩阵存储，文件以gzip格式压缩。
<strong>样本信息</strong> 1) MOVAS_IL1b_24h_48h_pooled [1355个细胞（列），31053个基因（行）]；2) MOVAS_serum_starved_control [1337个细胞（列），31053个基因（行）]
<strong>发表文献引用</strong> 待补充。