GSM7749225: CD8+ T cells VDJ seq, Lgr5-GFP+ mice; Mus musculus; RNA-Seq
10x scRNA-seq 5P single-cell RNA-seq and TCR-seq data from sorted colonic intraepithelial CD8+ T cells was processed with Cell Ranger 7.1.0 using the mm10 mouse genome indices from 10X Genomics. 7477 and 5332 cells were detected from Gfi1b and Lgr5 samples respectively. R (v4.2.1) was used for analysis and the packages Seurat (v4.3.0), DoubletFinder (v2.0.3), and SingleR (v1.10.0) were used to preprocess the data. Briefly, only cells with greater than 200 features and less than 5% mitochondrial reads were included. Further, the data was subset on predicted singlets as assigned by DoubletFinder and predicted T cells or NK cells as assigned by SingleR using the mouse bulk RNA-seq reference data (MouseRNAseqData) from celldex (v1.6.0). Cell cycle scoring was performed using Seurat's CellCycleScoring function. To specifically visualize the JEDI T cells, cells from both samples were merged and only cells belonging to that JEDI clonotype were analyzed. Traditional Seurat processing was performed: NormalizeData, FindVariableFeatures, ScaleData, RunPCA, RunUMAP, FindNeighbors, FindClusters. 2000 variable features were used and RunUMAP was performed with the top 30 dimensions. Clustering was performed with a resolution of 0.35. DEGs were calculated using the functions FindAllMarkers or FindMarkers for pairwise comparisons. For gene set enrichment analysis, escape (v1.6.0) was used whereby gene sets were customized to include canonical exhaustion, effector, tissue resident, or central memory genes. The AverageExpression function was performed to facilitate heat map creation. The package dittoSeq (v1.8.1) was also used to perform some visualizations. Monocle3 (v1.2.9) was used to infer pseudotime on non-proliferating cells using the package SeuratWrappers (v0.3.0) and setting those cells with highest Sell expression as the root node. Analysis of all bystander cells was performed in a similar fashion as above by excluding those cells with the JEDI T cell clonotype.