Single-cell RNA-seq of Mouse Infrarenal IVCs

Mouse infrarenal IVCs were collected and sequentially digested in two digestion buffers (PBS containing 200 U/ml collagenase I (SCR103, Sigma Aldrich), 0.05 U/ml elastase (E1250, Sigma Aldrich), 5 U/ml neutral protease (LS02111, Worthington), and 0.3 U/ml deoxyribonuclease I (M6101, Promega)^{50 (link)} for 20 min, followed by DMEM containing 5 mg/ml collagenase type II [C6885, Sigma-Aldrich] and 0.5 mg/ml elastase [LS002292, Worthington Biochemistry]) for 2.5 min at 37 °C). The tissue suspension was filtered with a 40μm cell strainer, then centrifuged at 500 g for 5 min. Cells were resuspended with PBS containing 0.04% BSA. Single-cell suspensions from 5 DVT-adjacent IVCs or 8 sham-IVCs were pooled together as one sample. 8000 cells per sample were loaded on a Chromium Controller (10x Genomics). The scRNA-seq libraries were constructed using the Chromium Single Cell 3′ v3.1 Reagent Kit according to the manufacturer’s guidelines (10x Genomics). cDNA libraries were uniquely sample indexed and pooled for sequencing. A MiSeq (Illumina) sequencing run was used to sample balance on a NovaSeq S1 flowcell (Illumina) using a 2×50 bp sequencing reaction targeting >90,000 reads/cell.