Pre-Analysis using FeaturePlot

1. load libraries

2. Load Seurat Object

#Load Seurat Object merged from cell lines and a control(PBMC) after filtration
SS_All_samples_Merged <- load("/home/bioinfo/0-imp_Robj/Harmony_integrated_All_samples_Merged_with_PBMC10x_with_harmony_clustering.Robj")

All_samples_Merged

An object of class Seurat 
64169 features across 59355 samples within 6 assays 
Active assay: SCT (27417 features, 3000 variable features)
 3 layers present: counts, data, scale.data
 5 other assays present: RNA, ADT, prediction.score.celltype.l1, prediction.score.celltype.l2, prediction.score.celltype.l3
 6 dimensional reductions calculated: integrated_dr, ref.umap, pca, umap, harmony, umap.harmony

DimPlot(All_samples_Merged, group.by = "cell_line", label = T, label.box = T)

3. Pre-Analysis using FeaturePlot

All_samples_Merged <- NormalizeData(All_samples_Merged, normalization.method = "CLR", margin = 1, assay = "ADT")

Normalizing across features

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FeaturePlot(All_samples_Merged, features = c("PRF1", "GZMB", "NKG7"), reduction = "umap.harmony")

FeaturePlot(All_samples_Merged, features = c("CCR7", "CCR4", "CD27", "CD62L", "SELL", "CD45RA", "CD45RO", "CD95"), reduction = "umap.harmony")

Warning: Could not find CD62L in the default search locations, found in ‘ADT’ assay insteadWarning: Could not find CD45RA in the default search locations, found in ‘ADT’ assay insteadWarning: Could not find CD45RO in the default search locations, found in ‘ADT’ assay insteadWarning: Could not find CD95 in the default search locations, found in ‘ADT’ assay instead

FeaturePlot(All_samples_Merged, features = c("IL7R", "CCR7", "CD45RO", "TCF7"), reduction = "umap.harmony")

Warning: Could not find CD45RO in the default search locations, found in ‘ADT’ assay instead

FeaturePlot(All_samples_Merged, features = c("KLHL42", "TOX", "DNM3", "PGM2L1", "SESN3"), reduction = "umap.harmony")

FeaturePlot(All_samples_Merged, features = c("CD3", "CD26", "CD7", "CD5", "CD70"), reduction = "umap.harmony")

Warning: Could not find CD3 in the default search locations, found in ‘ADT’ assay insteadWarning: Could not find CD26 in the default search locations, found in ‘ADT’ assay instead

FeaturePlot(All_samples_Merged, features = c("THEMIS", "LAIR1"), reduction = "umap.harmony")

FeaturePlot(All_samples_Merged, features = c("CD164", "CD274", "CD279", "KIR3DL2", "CD158k", "PD1" ), reduction = "umap.harmony")

Warning: Could not find PD1 in the default search locations, found in ‘ADT’ assay insteadWarning: The following requested variables were not found: CD279, CD158k

FeaturePlot(All_samples_Merged, features = c("CD183", "CD194", "CD196", "CCR10", "CD158k", "PD1" ), reduction = "umap.harmony")

Warning: Could not find PD1 in the default search locations, found in ‘ADT’ assay insteadWarning: The following requested variables were not found: CD183, CD194, CD196, CD158k

FeaturePlot(All_samples_Merged, features = c("BIRC3", "PIM2", "NPM1", "PRDX1", "DUSP4", "CCND2", "CCND3", "TNFRSF4" ), reduction = "umap.harmony")

FeaturePlot(All_samples_Merged, features = c("CD160", "TIGIT", "LAG3", "HAVCR2", "CTLA4", "PDCD1" ), reduction = "umap.harmony")

Save the Seurat object as an Robj file

# save(All_samples_Merged, file = "/home/bioinfo/0-imp_Robj/All_Samples_Merged_NormalizedADT_Margin2.Robj")