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README.md

Determining cellular heterogeneity in the human prostate with single-cell RNA sequencing

Data Analysis

  • Requirements:

    • 10x CellRanger analyzed data:
      • "filtered_gene_bc_matrice_mex" folder
      • csv file cellranger aggr used for aggregation
      • demultiplex csv file to define subsets of samples

  • Pipeline (r.scripts):

    • sc_Demultiplex (import 10x Cell Ranger data into R and subset samples based on user input)
    • sc_D-SampleReorder (reorder sample names)
    • sc_SeuratScore.CellCycle (identify cell cycle state)
    • sc_QC (filter cells, scale and remove variation associated to: UMI; % mitochondrial genes; S; G2M phase score, identify most variable genes and run PCA on them
    • sc_Cluster (perform tSNE and cluster using graph*based approach)
    • sc_PC.Score.Stress (PCA analysis of stress gene signature, projecction to PC1 used as "Stress Score", stressed cells and clusters removed cells re*clustered)
    • sc_QuSAGE.Lineage (clusters correlated to prostate population RNA-Seq DEGs of Epithelia and Fibromuscular Stroma to assign those identities to those clusters)
    • sc_LineageSubClust (separate identified Epithelia, and Stroma + Lineage Unknown cells, and recluster them independently)
    • sc_QuSAGE.EpiSubClust (Epithelial clusters correlated to DWS generated prostate population RNA-Seq DEGs of Basal, Luminal, and "Other" Epithelia, Lung Map generated Epithelial, C2 KEGG, and C5 GO BP genesets, to assign those identities to those clusters)
    • sc_QuSAGE.StSubClust (Stromal and Lineage Unknown clusters correlated to external DEGs of Endothelial Cells, Smooth Muscle Cells, and Fibroblasts, Lung Map generated Epithelial, C2 KEGG, and C5 GO BP genesets, to assign those identities to those clusters)
    • sc_MergeSubClust (merge identities of Eithelial and non-Epithelial clusters from QuSAGE)
    • sc_PC.Score.NE (PCA analysis of Neurocrine Epithelia protein marker signature, projecction to PC1 used as "NE Score", highest scoring cells identified as Neuroendocrine Cells)
    • sc_DEG (generate DEG lists between important cell types and predict surface and nuclear markers from them)
    • sc_Table (produce tables of population differences between samples)

  • Genesets:

    • "regev_lab_cell_cycle_genes.txt" G2M and S phase genes from Genome Res. 2015 Dec; 25(12): 1860–1872
    • "DEG_C2.CGP.M10970.txt" MSigDB C2 Chemical and Genetic Perturbations M10970 CHUANG_OXIDATIVE_STRESS_RESPONSE_UP
    • "DEG_Epi_2FC.txt" DWS generated DEGs of Epithelia from FACS population (bulk) RNA-sequencing
    • "DEG_FMSt_2FC.txt" DWS generated DEGs of Fibromuscular Stroma from FACS population (bulk) RNA-sequencing
    • "DEG_BE_2FC.txt" DWS generated DEGs of Basal Epithelia from FACS population (bulk) RNA-sequencing
    • "DEG_LE_2FC.txt" DWS generated DEGs of Luminal Epithelia from FACS population (bulk) RNA-sequencing
    • "DEG_OE_2FC.txt" DWS generated DEGs of "Other" Epithelia from FACS population (bulk) RNA-sequencing
    • "DEG_C5.BP.M11704.txt" MSigDB C5 GO Biological Processes M11704 GO_ENDOTHELIAL_CELL_DIFFERENTIATION
    • "DEG_C5.BP.M10794.txt" MSigDB C5 GO Biological Processes M10794 GO_SMOOTH_MUSCLE_CELL_DIFFERENTIATION
    • "DEG_C5.BP.M13024.txt" MSigDB C5 GO Biological Processes M13024 GO_REGULATION_OF_FIBROBLAST_PROLIFERATION
    • "EurUrol.2005.NE.txt" Neuroendocrine markers from Table 1 of Eur Urol. 2005 Feb;47(2):147-55
    • "Basal cells-signature-genes.csv" scRNA-Sequencing Lung Map generated top 20 DEGs for human lung Basal Cells
    • "Normal AT2 cells-signature-genes.csv" scRNA-Sequecing Lung Map generated top 20 DEGs for human lung Alveolar Type 2 Cells
    • "Club_Goblet cells-signature-genes.csv" scRNA-Sequencing Lung Map generated top 20 DEGs for human lung Club/Goblet Cells
    • "journal.pcbi.1004575.s026.XLSX" scRNA-Sequencing Lung Map generated DEGs for E16.5 mouse lung stromal subtypes (genes converted to human othologs with Ensembl):
      • Proliferative Fibroblasts
      • Myofibroblast/Smooth Muscle-like Cells
      • Pericytes, Matrix Fibroblasts
      • Endothelial Cells
      • Myeloid/Immune Cells
    • "c2.all.v6.1.symbols.gmt" MSigDB C2 Curated Gene Sets MSigDB C2
    • "c2.cp.kegg.v6.1.symbols" MSigDB C2 KEGG Gene Subsets KEGG
    • "c5.all.v6.1.symbols.gmt" MSigDB C5 Gene Ontology Gene Sets MSigDB C5
    • "c5.bp.v6.1.symbols.gmt" MSigDB C5 Gene Ontology Biological Processes Gene Subsets GO BP
    • "DWS.scStress.txt" DWS generated DEGs of Stressed Cells from scRNA-Sequencing
    • "DWS.scNE.txt" DWS generated DEGs of Neuroendocrine Cells from scRNA-Sequencing