Single-cell RNA-seq of post-ischemic hindlimb endothelial cells from endothelial specific Ppard knockout and wildtype mice

All of the animal experiments were approved by the institutional Animal Experimental Ethics Committee and were consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. All the mice were housed at 22 degree Celsius in a barrier facility and kept on a 12-hour light, 12-hour dark cycle with free access to food and water. The Ppard floxed mutant mice (B6.129S4-Ppardtm1Rev/J) and the VEC-cre transgenic mice (B6;129-Tg(Cdh5-cre)1Spe/J) were originally from Jackson laboratory. Both strains were backcrossed with C57BL/6 mice before they were crossed to generate endothelial cells specific deletion of Ppard as Ppardf/f;Cdh5Cre/+ (PpardEC-KO) mice; with their wild type controls Ppardf/f (PpardEC-WT) mice. Hindlimb ischemia (HLI) was induced by ligation of femoral artery. At 7 days after HLI surgery, gastrocnemius muscle from the injured leg was digested with 800U/ml Collagenase IV + 1U/ml Neutral Protease (both from Worthington Biochemical) for total 90 mins. The cells were then suspended in FACS buffer (2% FBS with 2 mmol/L EDTA in PBS), and filtered through 40-um strainer (BD Biosciences) to generate single-cell suspensions. Cells were firstly incubated with LIVE/DEAD Aqua (Thermo) for viability following the protocol of the manufacturer together with anti-CD16/CD32 (10 ug/mL, Biolegend) for 30 mins. Cells were then incubated with fluorescent-conjugated anti-mouse antibodies listed in Online Table I. Cells were fixed with 1.6% paraformaldehyde for 30 mins at 4 degree Celsius until further analysis using FACSAria Fusion (BD). Cells were then washed and stained with BV605 anti-CD45 (Biolegend, clone 30-F11, catalog # 103140), AlexaFluor 488 anti-CD31 (Biolegend, clone MEC13.3, catalog # 102514), APC anti-CD144 (BD, clone 11D4.1, catalog # 562242). CD45-CD31+CD144+ cells were sorted on FACSAria Fusion (BD) and resuspended in 0.4% BSA containing PBS. Data were analyzed using FlowJo. ScRNA-seq libraries were prepared using the Chromium Single Cell 3 prime Reagent Kits (v2 Chemistry) with the Chromium Controller (10x Genomics) as per the protocol of the manufacturer. The libraries were sequenced on an Illumina NovoSeq 6000 (Novogene).