Chromium single cell 3 v3.1 reagent kit

scRNAseq was done according to the manufacturer’s instructions (10x genomics) and as previously described⁸⁸ . Briefly, after quickly thawing the frozen BAL single-cell suspension in water bath, 2 × 10⁶ cells were taken for downstream processing. BAL single-cell suspensions were subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 3′ (v3.1) Reagent Kit in the BSL-3 laboratory as per manufacturer’s instructions (10x Genomics). Briefly, cell suspensions were loaded at 1000 cells/μL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier^{89 (link)} during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer’s instructions. Libraries were sequenced on a NovaSeq S4 (200 cycles) flow cell, targeting 30,000 read pairs/cell.

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.

scRNAseq was done according to the manufacturer instructions (10x genomics) and as previously described ^{77 (link)}. Briefly, after quickly thawing the frozen BAL single cell suspension in water bath, 2X10⁶ cells were taken for downstream processing. BAL single cell suspensions were subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 3’ (v3.1) Reagent Kit in the BSL-3 laboratory as per manufacturer’s instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/μL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier ^{78 (link)} during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer’s instructions. Libraries were sequenced on a NovaSeq S4 (200 cycle) flow cell, targeting 30,000 read pairs/cell.