GSM7156316: CJ_SKO911; Rattus norvegicus; RNA-Seq
We performed scRNA-seq of MA, AA and FA cells respectively from WKY/N and SHR/N rat of young and old group (male, 16-18 weeks old and 72-weeks old, 7-8 rats in each group). Only male rats were included to avoid the sex influence on experiment results. Rats were anaesthetized with intraperitoneal injection of 50 mg/kg sodium pentobarbital. The adequacy of anaesthesia throughout the procedure was confirmed by the lack of reflex response to toe stretch. Direct blood pressure was measured and recorded with a heparinized fluid-filled catheter that was inserted into the isolated femoral artery. While still being under 50 mg/kg intraperitoneal sodium pentobarbital anaesthesia, the rats were sacrificed by exsanguination via transcardial perfusion with 50 mL phosphate buffered saline (PBS) and subsequent heart excision. All branches of superior and inferior MAs (excluding the superior and inferior MAs themselves), AA (thoracic artery and abdominal artery; aortic arch is not included) and FA (Below the iliac artery branch) were collected and perivascular tissues were carefully cleaned. Pooled tissue from each group and artery type, respectively, was obtained. As described before(Gu et al., 2019), the arteries were digested with 2 mg/mL collagenase I (Invitrogen; 17018-029) and 2 mg/mL dispase II (Sigma; D4693) in Hank's balanced salt solution (with calcium and magnesium) at 37 ℃ for 30 minutes on a shaker at the speed of 100 rpm. Digested mix was passed through 40 μm strainer and stained with LIVE/DEAD Fixable Near-IR (APC/Cy7 channel) Dead Cell Stain Kit (Invitrogen; L34975, 1:1000) and Hoechst 33342 (Invitrogen; H3570, 1:1000) for 20 minutes. Unstained cells and single stained cells served as control. After 1 wash in PBS for 5 minutes, cells resuspended in PBS were sorted with FACS Aria II (BD Biosciences). Single nucleated live cells (Hoechst+/APC/Cy7-population) were sorted into PBS with 0.04% BSA. After centrifugation at 300 g for 5 min, the cells were loaded to 10x Genomics Chromium chip following manufacturer's recommendations with target capture of 6,000 cells. Standard 10x Chromium Single Cell 3′ v2 (10x Genomics) protocols were followed for scRNA-seq for reverse transcription and library preparation. The generated scRNA-seq library was sequenced with Nova PE150 with the sequencing depth of 30 million per cell. RNA libraries were prepared for sequencing using standard Illumina protocols