Murine lymph node FRCs during development

For stromal cell isolation from peripheral (inguinal, brachial and axillary) and mesenteric lymph nodes, tissues were disrupted into small pieces using small needles and collected in RPMI 1640 medium containing 2% FCS, 20 mM HEPES pH 7.2 (Lonza), 0.16 mg/ml collagenase P, Dispase 30 μg/ml (Roche) and 25 μg/ml DNase I. The dissociated tissue was incubated for 45 min at 37 °C, during the incubation time the tissue was resuspended and supernatant was collected every 15 min. Enrichment of stromal cells was performed by incubating the cell suspension with MACS anti-CD45 and anti-TER119 microbeads (Miltenyi Biotec) and passing it through MACS LS columns (Miltenyi Biotec). Single-cell suspensions were stained for further cell sorting. Stromal cells from peripheral and mesenteric lymph nodes of Ccl19-iEYFP and Cxcl13-EYFP mice were sorted for CD45^– TER119^– CD31^– stromal cells using a BD FACSMelody cell sorter (BD Biosciences).The generation of the cDNA library was performed following the established commercial protocol for Chromium Single Cell 3’ Reagent Kit (v3.1 Chemistry). Subsequent sequencing was performed on a Illumina NovaSeq 6000 (Functional Genomic Center Zurich). CellRanger was used for gene expression analysis from the sequencing output (v5.0.1 and v.8.0.1) with the Ensembl GRCm38.9 and GRCm38.102 release as reference. Quality control included the removal of cells with very high or low UMI counts (&gt;2.5 median absolute deviation from the median of all cells), very high or low total number of detected genes (&gt;2.5 median absolute deviation from the median of all cells), and elevated mitochondrial gene content (&gt;2.5 median absolute deviations above the median of all cells) using R v.4.2.1 with the R/Bioconductor package scater (v.1.24.0). Cells expressing B cell, T cell or neuronal cell markers (<i>Ptprc</i>, <i>Cd3e</i>, <i>Cd79a</i>, <i>L1cam</i>) were excluded prior further analysis.Downstream analysis was performed using the Seurat R package (v.4.3.0 and v.4.4.0) and included procedures such as data normalization and integration for collective assignment of adult FRC subsets and VSMCs across peripheral and mesenteric lymph nodes, data scaling, dimensionality reduction through PCA and UMAP, graph-based clustering, and the detection of unbiased cluster markers. FRC and VSMC clusters from lymph nodes of adult mice were assigned according to their expression profiles of both unbiased cluster markers and canonical marker genes described in previous studies. Differentially expressed gene analysis between lymph node entities and conditions was performed using the <i>FindConservedMarkers</i> and <i>FindMarkers</i> functions as implemented in the Seurat R package (v.4.3.0 and v.4.4.0). Significantly enriched GO terms were determined using the enrichGO function from the clusterProfiler R/Bioconductor package (v.4.4.4).Differentiation trajectory pathways of Ccl19-iEYFP⁺ and Cxcl13-EYFP⁺ cells from mesenteric lymph nodes were modeled using the slingshot R package (v.2.8.0). For trajectory inference of adult FRC and VSMC clusters were set as end points, whereas the cluster with cells from lymph node anlagen at E18 was used as starting point. The fitGAM function of the tradeSeq R/Bioconductor package (v.1.14.0) was used to model the expression of the 2000 most variable genes along the inferred differentiation lineages. The average activity of transcription factors in clusters of Ccl19-iEYFP⁺ cells from mesenteric lymph nodes was inferred using AUCell by the run_aucell function as implemented in the decoupleR R package (v.2.6.0).