Genomics controller

Approximately 10,000 viable enriched stromal cells were loaded on a 10x Genomics controller (10x Genomics, San Francisco, CA) to generate barcoded single-cell Gel Bead in-Emulsions (GEMs) using the 10x Genomics 3′ kit as previously described.³¹ (link) Complementary DNA libraries were sequenced using a NovaSeq 6000 (Illumina, San Diego, CA) at the University of Colorado Genomics Shared Resource Core. To control for batch effects 3 independent sequencing experiments were performed which included both control- and HFHC diet–fed mice. Quantification and analysis of single cell sequencing was performed using the R packages Seurat and scran as previously described.³¹ (link) Ridge plots were generated using Seurat’s RidgePlot function. Cells were classified and correlation plots were generated using clustifyr.⁵⁵ (link) For cell cycle analysis, Seurat assigns each cell a score based on its expression of G2/M and S phase markers. These marker sets are anti-correlated in their expression levels, and cells expressing neither are assigned to G1 phase. Cells with fewer than 250 detectable genes or >20% of unique molecular identifiers (UMIs) derived from mitochondrial genes were excluded from the analysis to eliminate cells with insufficient expression data for clustering and dead cells, respectively.