Endothelial cell enrichment and quantitative PCR (qPCR)

Single-cell suspensions were prepared from liver, lung, kidney, brain, and DRGs by enzymatic digestion at 37°C using the following enzymes and incubation times. Liver, small piece of one lobe (1 mg/ml Collagenase I 17100-017; Gibco, 1 mg/ml; Collagenase II {"type":"entrez-nucleotide","attrs":{"text":"LS004176","term_id":"1321650548","term_text":"LS004176"}}LS004176; Worthington, 5 mg/ml Dispase II D4693; Sigma-Aldrich, and 7.5 μg/ml DNAse I 11284932001, 30 min; Roche), lung, one lobe (1 mg/ml; Collagenase II, 2.5 mg/ml Collagenase IV {"type":"entrez-nucleotide","attrs":{"text":"LS004188","term_id":"1321650536","term_text":"LS004188"}}LS004188; Worthington, and 15 μg/ml DNAse I, 50 min), kidney, one (2 mg/ml; Collagenase I and 7.5 μg/ml DNAse I, 45 min), forebrain (2 mg/ml; Collagenase I, 0.5 mg/ml DNAse I, 30 min), and DRGs, ∼40 (2 mg/ml; Collagenase I, 5 mg/ml Dispase II, 0.5 mg/ml DNAse I, 45 min). Single-cell suspensions were resuspended in FACS buffer and labeled with CD31 microbeads (130-097-418; Miltenyi) and enriched on MS columns (130-042-201; Miltenyi) according to the manufacturer’s instructions. Cells were pelleted and lysed in RLT buffer. RNA was isolated using the RNeasy Micro Kit (74004; Qiagen) and reverse transcribed using iScript cDNA synthesis kit (1708890; Bio-Rad) according to manufacturer instructions. qPCR was performed on a C1000 Touch thermal cycler equipped with a CFX384 detection module (Bio-Rad) with SYBR Green Master Mix reaction (4367659; Bio-Rad) and the following PCR primer sequences (Sigma-Aldrich): Gapdh (F: 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′, R: 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′), Pecam1 (F: 5′-ACG​CTG​GTG​CTC​TAT​GCA​AG-3′, R: 5′-TCA​GTT​GCT​GCC​CAT​TCA​TCA-3′), Slc2a1 (F: 5′-CAG​TTC​GGC​TAT​AAC​ACT​GGT​G-3′, R: 5′-GCC​CCC​GAC​AGA​GAA​GAT​G-3′), Slc7a5 (F: 5′-CTT​CGG​CTC​TGT​CAA​TGG​GT-3′, R: 5′-TTC​ACC​TTG​ATG​GGA​CGC​TC-3′), Mfsd2a (F: 5′-AAA​GAC​ACG​CAA​AAT​GCT​TAC​CT-3′, R: 5′-AAT​GAA​GGC​ACA​GAG​GAC​GTA​GA-3′), Gpihbp1 (F: 5′-AGG​GCT​GTC​CTC​CTG​ATC​TTG-3′, R: 5′-GGG​TCC​GCA​TCA​CCA​TCT​T-3′), Slco2a1 (F: 5′-ATT​AAG​GTC​TTC​GTG​CTT​TGT​CA-3′, R: 5′-GTA​GGC​ACT​GTA​GAG​CAA​CTG-3′).