Single-cell RNA-seq of eWAT stromal vascular fraction cells from WT and Camk2d myeloid conditional knockout mice after high-fat diet
Epididymal white adipose tissue (eWAT) stromal vascular fraction (SVF) cells were profiled by 10x Genomics Chromium Single Cell 3' v1 scRNA-seq to characterize immune-cell states in diet-induced obesity. SVF cells were isolated by collagenase digestion, filtered, and processed when viability exceeded 85%. Libraries were sequenced on an Illumina NovaSeq 6000 (PE150). Reads were aligned to mm10 and gene–cell count matrices were generated with Cell Ranger count v5.0.0 and aggregated across samples with Cell Ranger aggr. Downstream analyses were performed in Seurat v4.1.1 with QC filtering, ambient RNA correction (SoupX v1.3.0), doublet detection (DoubletFinder v2.0.2), batch correction (fastMNN), clustering/UMAP, and differential expression testing (MAST). Cell types were annotated using SingleR v1.4.1 and canonical markers; non-immune cells were excluded for immune-focused analyses. Additional analyses included pseudotime inference (Monocle2 v2.14.0) and cell–cell communication analysis (CellChat v0.0.2). Overall design: Two experimental groups (WT vs Camk2d myeloid conditional knockout) were compared after 10 weeks of high-fat diet. For each biological replicate, eWAT SVF was dissociated and ~10,000–13,000 cells were loaded per Chromium channel to generate one scRNA-seq library per sample.