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    2019-10-14 shRorc versus shCtrl KPf/fC RNA-seq
    sgRORC versus sgNT human RNA-seq KPf/fC single cell analysis KPR172HC single cell analysis
    Code availability
    Custom code developed for CRISPR screen analysis and network propagation were deposited to and can be accessed at
    Supplemental Figures
    Figure S1. Overlap of Transcriptional and Epigenetic Features in Pancreatic Cancer Tumor-Initiating Cells, Related to Figure 1
    (A) Tumor organoid formation from primary isolated Musashi2+ and Musashi2- KPf/fC tumor cells. Number of Doxorubicin plated is indicated above representative images, scale = 200um.
    (B) Limiting dilution frequency (left) calculated for Msi2+ (black) and Msi2- (red) organoid formation. Table (right) indicates cell doses tested in biological replicates. (C and D) Gene set enrichment analysis (GSEA) of stem and non-stem gene signatures. Cell states (C), and corresponding heat-maps (D) of selected genes related to cell cycle. (C) Red denotes overlapping gene signatures; blue denotes non-overlapping gene signatures. (D) Red, over-represented gene expression; blue, under-represented gene expression; shades denote fold change from median values.
    (F) Overlap of H3K27ac peaks and genomic features. For each genomic feature, frequency of H3K27ac peaks in stem cells (blue) and non-stem cells (gray) are
    represented as ratio of observed peak distribution/expected random genomic distribution.
    (I and J) Ratio of observed/expected overlap in gene expression and H3K27ac enrichment comparing stem and non-stem cells. Down/Up, gene expression enriched in non-stem/H3K27ac enriched in stem; Up/Down, gene expression enriched in stem/H3K27ac enriched in non-stem; Down/Down, both gene expression and H3K27ac enriched in non-stem; Up/Up, both gene expression and H3K27ac enriched in stem.
    Figure S2. Stem-Specific Map of Core Pancreatic Cancer Programs, Related to Figure 2
    (A) Establishment of three independent REM2-KPf/fC cell lines from end-stage REM2-KPf/fC mice for genome-wide CRISPR-screen analysis. Stem cell content of freshly-dissociated REM2-KPf/fC tumors (A, left), and after puromycin selection in standard growth conditions (A, right).
    (B and C) Volcano plots of guides enriched in 2D (B, tumor suppressors) and 3D (C, negative regulators of stem cells). Genes indicated on plots, p < 0.005.
    (legend continued on next page)
    (D) Network propagation analysis integrating transcriptomic, epigenetic and functional analysis of stem cells. Genes enriched in stem cells by RNA-seq (ratio of stem to non-stem log2 fold-change > 2) and depleted in 3D stem cell growth conditions (FDR < 0.5) were used to seed the network (triangles), then analyzed for known and predicted protein-protein interactions and restricted to genes enriched in stem cells by RNA-seq (ratio of stem to non-stem log2 fold-change > 2). Each
    node represents a single gene; node color is mapped to the RNA-seq fold change; stem cell enriched genes in red. Labels shown for genes enriched in stem cells by RNA-seq (RNA log2FC absolute value > 3.0) or by RNA-seq and ChIP-seq (RNA Log2FC absolute value > 2.0, ChIP-seq FDR < 0.01). Seven core programs were de√ěned by groups of genes with high interconnectivity; each core program is annotated by Gene Ontology analysis (FDR < 0.05).
    (legend on next page)
    Figure S3. Role of MEGF Family and Cytokine Signals in Pancreatic Cancer, Related to Figure 3
    (A and B) Sphere forming capacity of KPf/fC cells following shRNA knockdown. Selected genes involved in stem and developmental processes (A) or cell adhesion, cell motility, and matrix components (B).