I've made available some publicly available data containing single cell RNA-Seq data for 5k immune cells as well as a script that runs the Seurat workflow to define cell-type clusters in this data.

You can get the data, R script and results from stampede2 here:

/work2/projects/BioITeam/projects/courses/rnaseq_course/day_5_single_cell_data
	Script: seuratTotalScript5k.mod.R
	Data (after preprocessing by cellRanger): filtered_feature_bc_matrix
	Results generated: results

Unfortunately, the packages required to run Seurat on TACC  are either out-of-date or not installed. So, we are not going to run this script today.

The script does the following:

• Load Data and Create Seurat Object

  pbmc_data <- Read10X(data.dir = "filtered_feature_bc_matrix") pbmc <- CreateSeuratObject(counts = pbmc_data)

  
  

• Normalize Data Using SCTransform and Regress out Genes Related to Cell Cycle

  s.genes <- cc.genes$s.genes g2m.genes <- cc.genes$g2m.genes pbmc <- CellCycleScoring(pbmc, s.features = s.genes, g2m.features = g2m.genes, set.ident = TRUE) pbmc$CC.Difference <- pbmc$S.Score - pbmc$G2M.Score pbmc <- SCTransform(pbmc, vars.to.regress = "CC.Difference")

  
  

• Identify Highly Variable Features

  pbmc <- FindVariableFeatures(pbmc, selection.method = "vst", nfeatures = 2000) top10 <- head(VariableFeatures(pbmc), 10) pdf("variableFeaturesPlot.pdf") plot1 <- VariableFeaturePlot(pbmc) plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE) CombinePlots(plots = list(plot1, plot2)) dev.off()

  
  

• Scale the Data

  all.genes <- rownames(pbmc) pbmc <- ScaleData(pbmc, features = all.genes)

  
  

• Select Dimensions using PCA (using variable features)

  #Perform PCA for Dimensionality Reduction pbmc <- RunPCA(pbmc, features = VariableFeatures(object = pbmc)) #Print and Examine PCA Results in Multiple Formats pdf("findDimensions.pdf") print(pbmc[["pca"]], dims = 1:5, nfeatures = 5) VizDimLoadings(pbmc, dims = 1:2, reduction = "pca") DimPlot(pbmc, reduction = "pca") #Generate Principal Component Heatmaps through PC_30 DimHeatmap(pbmc, dims = 1, cells = 500, balanced = TRUE) DimHeatmap(pbmc, dims = 1:15, cells = 500, balanced = TRUE) DimHeatmap(pbmc, dims = 16:30, cells = 500, balanced = TRUE) #Create Elbow Plot of Principal Components through PC_30 ElbowPlot(pbmc, ndims = 30, reduction = "pca") dev.off()

  
  

• Cluster the cells (using the selected number of dimensions)

  #Cluster the Cells for the selected numeber of Principal Components (we selected 14 for 5k Data) pbmc <- FindNeighbors(pbmc, dims = 1:14) pbmc <- FindClusters(pbmc, resolution = 0.5) #View the Cluster IDs of the First 5 Cells head(Idents(pbmc), 5)

  
  

• Visualize the clusters using tSNE or UMAP

  pbmcTSNE <- RunTSNE(pbmc, dims = 1:14) DimPlot(pbmcTSNE, reduction = "tsne")

  
  

• Find markers for each cluster

  pbmc.markers <- FindAllMarkers(pbmcTSNE, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25) pbmc.markers %>% group_by(cluster) %>% top_n(n = 2, wt = avg_logFC) write.csv(pbmc.markers, file = "markersAll.csv") #Generate Heatmap of Top 10 Markers per Cluster top10 <- pbmc.markers %>% group_by(cluster) %>% top_n(n = 10, wt = avg_logFC) DoHeatmap(pbmcTSNE, features = top10$gene, size = 3) + theme(axis.text.y = element_text(size = 5)) + NoLegend()

  
  

• Identify cells expressing genes of interest 

  markerGenes <- c("CD27", "CCR7", "CD8A", "CD8B", "IL7R", "GZMK", "CD79A", "CD37", "CD160", "NKG7", "GNL$ seuratClusters <- function(output, genes) { for (gene in genes) { print(output[output$gene == gene,]) } } seuratClusters(pbmc.markers, markerGenes) #Visualize Feature Expression on TSNE Plot of Top Gene per Cluster FeaturePlot(pbmcTSNE, features = c("LTB","S100A8","CCL5","NOSIP")) FeaturePlot(pbmcTSNE, features = c("LINC02446","IGKC","GNLY","MT-CO3")) FeaturePlot(pbmcTSNE, features = c("CST3","NKG7","KLRB1","LST1")) FeaturePlot(pbmcTSNE, features = c("PPBP","AC084033.3")) #Visualize Expression Probability Distribution Across Clusters for Top Gene per Cluster VlnPlot(pbmcTSNE, features = c("LTB","S100A8","CCL5","NOSIP")) VlnPlot(pbmcTSNE, features = c("LINC02446","IGKC","GNLY","MT-CO3")) VlnPlot(pbmcTSNE, features = c("CST3","NKG7","KLRB1","LST1")) VlnPlot(pbmcTSNE, features = c("PPBP","AC084033.3"))

  
  

• Label clusters as cell types based on expression of known marker genes.

new.cluster.ids <- c("CD4+ Memory Cells", "CD16+ and CD14+ Monocytes", "Regulatory T Cells", "CD8+ T Ce$
names(new.cluster.ids) <- levels(pbmcTSNE)
pbmcTSNE <- RenameIdents(pbmcTSNE, new.cluster.ids)
DimPlot(pbmcTSNE, reduction = "tsne", label = TRUE)