title: GSM7256740: CTR, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: After removing the perivascular adipose and connective tissues, the isolated carotid arteries were cut into 0.5 mm2 pieces. Then, the diced vascular tissues were digested in enzyme solution (2 mg/mL collagenase II, 1mg/ml DNase I) in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum (FBS) and pipetted for 5 - 10 times. The cell suspension was filtered by passing through 30 μm stacked cell strainer and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100 μl 1× PBS (0.04% BSA), and added with 1× red blood cell lysis buffer (MACS 130 - 094 - 183, 10×) and incubated for 2 - 10 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300g for 5 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads (MACS 130 - 090 - 101) and remove dead cells using Miltenyi® Dead Cell Removal Kit (MACS 130 - 090 - 101). Then the suspension was resuspended in 1× PBS (0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C. The cell pellet was resuspended in 1× PBS (0.04% BSA). The overall cell viability was more than 90% confirmed by trypan blue exclusion. The single cell suspensions were counted using Countess II Automated Cell Counter and the concentration was adjusted to 700 - 1200 cells / μl. Single-cell suspensions were loaded to 10× Chromium to capture 10,000 single cells using a 10× Genomics Chromium Single-Cell 3′ reagent kit (V3) according to the manufacturer′s instructions (10× Genomics, CA). The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150bp) by LC-Bio Technology Co., Ltd (Hangzhou, China) at a minimum depth of 20,000 reads per cell.