## ----import_libraries--------------------------------------------------------- library("Nebulosa") library("Seurat") library("BiocFileCache") ## ----download_and_untar_file-------------------------------------------------- bfc <- BiocFileCache(ask = FALSE) data_file <- bfcrpath(bfc, file.path( "https://s3-us-west-2.amazonaws.com/10x.files/samples/cell", "pbmc3k", "pbmc3k_filtered_gene_bc_matrices.tar.gz" )) untar(data_file, exdir = tempdir()) ## ----read_data---------------------------------------------------------------- data <- Read10X(data.dir = file.path(tempdir(), "filtered_gene_bc_matrices", "hg19" )) ## ----create_seurat_object----------------------------------------------------- pbmc <- CreateSeuratObject( counts = data, project = "pbmc3k", min.cells = 3, min.features = 200 ) ## ----qc----------------------------------------------------------------------- pbmc[["percent.mt"]] <- PercentageFeatureSet(pbmc, pattern = "^MT-") pbmc <- subset(pbmc, subset = nFeature_RNA < 2500 & percent.mt < 5) ## ----norm, message=FALSE, warning=FALSE--------------------------------------- pbmc <- SCTransform(pbmc, verbose = FALSE) ## ----dim_red, message=FALSE, warning=FALSE------------------------------------ pbmc <- RunPCA(pbmc) pbmc <- RunUMAP(pbmc, dims = 1:30) ## ----clustering, message=FALSE, warning=FALSE--------------------------------- pbmc <- FindNeighbors(pbmc, dims = 1:30) pbmc <- FindClusters(pbmc) ## ----plot_cd4----------------------------------------------------------------- plot_density(pbmc, "CD4") ## ----cd4_comparison----------------------------------------------------------- FeaturePlot(pbmc, "CD4") FeaturePlot(pbmc, "CD4", order = TRUE) ## ----cd4_and_clustering------------------------------------------------------- DimPlot(pbmc, label = TRUE, repel = TRUE) ## ----plot_cd3d---------------------------------------------------------------- plot_density(pbmc, "CD3D") ## ----fig.height=10------------------------------------------------------------ p3 <- plot_density(pbmc, c("CD8A", "CCR7")) p3 + plot_layout(ncol = 1) ## ----fig.height=14------------------------------------------------------------ p4 <- plot_density(pbmc, c("CD8A", "CCR7"), joint = TRUE) p4 + plot_layout(ncol = 1) ## ----------------------------------------------------------------------------- p_list <- plot_density(pbmc, c("CD8A", "CCR7"), joint = TRUE, combine = FALSE) p_list[[length(p_list)]] ## ----fig.height=14------------------------------------------------------------ p4 <- plot_density(pbmc, c("CD4", "CCR7"), joint = TRUE) p4 + plot_layout(ncol = 1) ## ----fig.height=10------------------------------------------------------------ p4[[3]] / DimPlot(pbmc, label = TRUE, repel = TRUE)