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

• Contact: Gervaise H. Henry
• Email: gervaise.henry@utsouthwestern.edu
• Institution: UT Southwestern Medical Center
• Department: Urology

Data Analysis

• Requirements:

  • 10x CellRanger analyzed data:
    • "filtered_gene_bc_matrice_mex" folder
    • csv file cellranger aggr used for aggrigation
    • 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_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_QuSAGE.Epi (Epithelial clusters correlated to prostate population RNA-Seq DEGs of Basal, Luminal, and "Other" Epithelia to assign those identities to those clusters)
  • sc_QuSAGE.St (Stromal clusters correlated to external DEGs of Endothelial Cells, Smooth Muscle Cells, and Fibroblasts to assign those identities to those clusters)
  • 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_QuSAGE.LGEA.Epi (Epithelial clusters correlated to top 20 DEGs from scRNA-seq of human lung Basal Cells, Alveolar Type 2 Cells, Club/Goblet Cells to determine similarity to lung epithelial cell types)
  • sc_QuSAGE.LGEA.St (Stromal clusters correlated to human orthologs of mouse lung stromal DEGs from scRNA*seq to determine similarity to:)
    • Proliferative Fibrobblasts
    • Myofibroblast/Smooth Muscle-Like Cells
    • Matrix Fibroblasts
    • Endothelial Cells
    • Myeloid/Immune 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
  • "DWS.scStress.txt" DWS generated DEGs of Stressed Cells from scRNA-Sequencing
  • "DWS.scNE.txt" DWS generated DEGs of Neuroendocrine Cells from scRNA-Sequencing