vessel_size_share.gz.h5ad

Sample preparation- At birth, neonatal C57BL-6 (Charles River) mice were housed in room air (normoxia; N) or 80% O2 (hyperoxia; H) for 72hr before euthanasia (N=6 per exposure; 3M 3F). Lungs were perfused with HBSS, excised, finely minced, pooled, and digested in Liberase TM (0.2mg/mL) plus DNaseI (0.01mg/mL) for 10min at 37.5 in a bacteriological shaker. The suspension was triturated 15 times. Liberase was inactivated using 2mM EDTA and cold FBS prior to pelleting the cell suspension. Cells were treated with 1X RBC Lysis for 5min then passed over a 30μm cell strainer before microbead enrichment (SmartStrainer, Miltenyi). In a parallel set of experiments, lungs were pressure fixed at 20cm H2O and paraffin embedded (FFPE) for in situ validation experiments.
Enrichment by magnetic associated cell sorting (MACS)- Immune cells were depleted using anti-CD45 coated Dynabeads (Invitrogen). The supernatant was pelleted, counted, and resuspended with blocking buffer (anti-mouse CD16/CD32 and anti-rat IgG; 1:100) for 10min. Mouse anti-BST1–APC (PVEC marker, Biolegend) and rat anti-CD31–biotin (pan-EC marker, BD Pharminogen) antibodies were added to the cells (2μg/10M cells) and incubated on a HulaMixer for 20 minutes. To ensure sufficient PVEC for our analysis, following the manufacturer’s protocol, cells were first enriched for BST1 using the MACS anti-APC microbeads and then the flow through was enriched for CD31 using anti-biotin microbeads (Miltenyi MACS).
Chromium GEMX 3’ v4 gene expression- Following the manufacturers protocol, enriched populations were then adjusted to 1500 cell/μL to obtain the 20,000 cell target input for gel bead emulsion formation (10X Genomics). Samples that met QC standards (Bioanalyzer, Stanford Protein and Nucleic Acid core) were used for barcoding, library construction and sequenced by NovoSeq Χ at a depth of 1B paired reads per sample (NovoGene Corporation Inc.).