add script

Figures_scRNA_seq.R

+########################
+## Single cell analysis
+########################
+library(Seurat)
+library(clusterProfiler)
+library(patchwork)
+library(ggplot2)
+library(RColorBrewer)
+library(scales)
+library(grid)
+library(gridExtra)
+
+## full data all the condition 
+readRDS('full_csc_2023.rds')
+
+## senescence lists
+gmt.obj <- read.gmt('senescence_list_paper.gmt')
+gmt.obj$term <- factor(gmt.obj$term)
+table(gmt.obj$term)
+
+ll <- unique(gmt.obj$term) 
+
+
+full_obj$CSC_clusters <- ifelse(full_obj$CSC_celltype == 'HSC_0', 'HSC-enriched_1',
+                                ifelse(full_obj$CSC_celltype == 'HSC_1', 'HSC-enriched_1',
+                                       ifelse(full_obj$CSC_celltype == 'HSC_2', 'HSC-enriched_2',
+                                              ifelse(full_obj$CSC_celltype == 'HSC_3', 'HSC-enriched_1',
+                                                     ifelse(full_obj$CSC_celltype == '1', 'Myeloid-biased_prog',
+                                                            ifelse(full_obj$CSC_celltype == '3',  'Erythroid-biased_prog',
+                                                                   ifelse(full_obj$CSC_celltype == '4', 'Neutrophil-biased_prog',
+                                                                          ifelse(full_obj$CSC_celltype == '5', 'PreB/Mono/DC_prog','unknown'
+                                                                          ))))))))
+
+
+
+
+full_obj$CSC_clusters <- factor(full_obj$CSC_clusters, levels=c('HSC-enriched_1', 'HSC-enriched_2',
+                                                                'PreB/Mono/DC_prog', 'Myeloid-biased_prog',
+                                                                'Neutrophil-biased_prog','Erythroid-biased_prog'
+))
+
+
+table( colnames(mix3_obj_new) == colnames(full_obj))
+full_obj$neworignames <- ifelse(full_obj$orig.ident == 'RNPneg', 'RNP NEG', 
+                                ifelse(full_obj$orig.ident == 'GTGFPneg', 'GFP-',
+                                       ifelse(full_obj$orig.ident == 'GTGFPpos', 'GFP+', 
+                                              ifelse(full_obj$orig.ident == 'RNPhs', 'RNP HS',full_obj$orig.ident)))))
+
+
+Idents(full_obj) <- 'neworignames'
+full_obj <- subset(full_obj, idents = c("GFP+", "GFP-","RNP NEG", "RNP HS"))
+full_obj$neworignames <- factor(full_obj$neworignames, levels =c("RNP NEG","RNP HS",
+                                                                           "GFP-","GFP+"))
+
+Idents(full_obj) <- 'CSC_clusters'
+for (l in 1:length(ll)) {
+  group <-ll[l] 
+  nn <- gsub('\\)','',gsub('\\(','',gsub(' ','_',group)))
+  nn <- gsub('-','_',nn)
+  glist <- gmt.obj[gmt.obj$term == group,'gene'] 
+  gmt_features <- list(glist)
+  full_obj <- AddModuleScore(
+    object = full_obj,
+    features = gmt_features, ## 
+    name = nn, 
+    
+  )}
+
+
+
+clcol <- c("RNP NEG"="black",  "RNP HS"="gray36", "GFP-"="grey", "GFP+"="green3")
+Idents(full_obj) <- 'CSC_clusters'
+
+p <- list()
+sasp <- list()
+for (n in levels(full_obj$CSC_clusters)){
+  sub <- subset(full_obj, idents = n)
+  p[[n]] <-  VlnPlot(object = sub, features = c("FRIDMAN_SENESCENCE_UP1"), 
+                     group.by  ="neworignames", col = clcol,  pt.size = 0,sort = F)+
+    geom_boxplot(width=0.2,outlier.shape = NA)+ theme(legend.position="none",
+                                                      axis.title.x = element_blank())+
+    ggtitle(paste0(gsub('_', ' ',n)))
+  sasp[[n]] <- VlnPlot(object = sub, features = c("SASP_SCHLEICH1"), 
+                       group.by  ="neworignames", col = clcol,  pt.size = 0,sort = F)+
+    geom_boxplot(width=0.2,outlier.shape = NA)+ theme(legend.position="none",
+                                                      axis.title.x = element_blank())+
+    ggtitle(paste0(gsub('_', ' ',n)))
+}
+
+## get legend just from one 
+lg_legend<-function(a.gplot){
+  tmp <- ggplot_gtable(ggplot_build(a.gplot))
+  leg <- which(sapply(tmp$grobs, function(x) x$name) == "guide-box")
+  legend <- tmp$grobs[[leg]]
+  return(legend)}
+
+mylegend<- lg_legend(VlnPlot(object = sub, features = c("FRIDMAN_SENESCENCE_UP1"),
+                             group.by  ="neworignames", col = clcol,  pt.size = 0,sort = F)+
+                       geom_boxplot(width=0.2,outlier.shape = NA))
+
+
+svg("senescence_violin.svg", width =20, height = 4, pointsize=12)
+grid.arrange(p$`HSC-enriched_1`, p$`HSC-enriched_2`, p$`PreB/Mono/DC_prog`, 
+             p$`Myeloid-biased_prog`, p$`Neutrophil-biased_prog`, p$`Erythroid-biased_prog`, mylegend, 
+             nrow = 1,top = textGrob("FRIDMAN SENESCENCE UP",gp=gpar(fontsize=20,font=3)))
+dev.off()
+
+svg("sasp_violin.svg", width =20, height = 4, pointsize=12)
+grid.arrange(sasp$`HSC-enriched_1`, sasp$`HSC-enriched_2`, sasp$`PreB/Mono/DC_prog`, 
+             sasp$`Myeloid-biased_prog`, sasp$`Neutrophil-biased_prog`, sasp$`Erythroid-biased_prog`, mylegend, 
+             nrow = 1,top = textGrob("SASP SCHLEICH",gp=gpar(fontsize=20,font=3)))
+dev.off()
+
+
+## RIDGE plot 
+p <- RidgePlot(object = full_obj, features = c("FRIDMAN_SENESCENCE_UP1"),group.by = "neworignames", cols = clcol)+ theme(legend.position="none",
+                                                                                                                         axis.title.x = element_blank(),
+                                                                                                                axis.title.y = element_blank())
+svg("senescence_ridge.svg", width =11, height = 6, pointsize=12)
+p + ggtitle("FRIDMAN SENESCENCE UP")
+dev.off()
+
+## final sasp
+p <- RidgePlot(object = full_obj, features = c("SASP_SCHLEICH1"),group.by = "neworignames", cols = clcol)+ theme(legend.position="none",
+                                                                                                                 axis.title.x = element_blank(),
+                                                                                                                 axis.title.y = element_blank())
+svg("sasp_SCHLEICH_ridge.svg", width =11, height = 6, pointsize=12)
+p + ggtitle("SASP SCHLEICH")
+dev.off()
+