Extended Data Fig. 1: Identification of the gene expression program of GSCs.

a, Single-cell differentiation trajectory of GBM cells reconstructed by Monocle2 using single-cell RNA-seq data of the indicated cell populations from primary GBM samples of the Darmanis dataset. b, Gene set enrichment analysis (GSEA) for association between the cell populations at the start and at the end of the pseudotime trajectory of the neoplastic cells of the Darmanis datasets (as depicted in Fig. 1c), and gene sets denoting the identity of specific cell types. Gene lists denoting early neural progenitor cells (RG: Radial Glia; oRG: outer Radial Glia; vRG: ventricular Radial Glia) or neural stem cells (NSC) are indicated in red; those identifying neurons, astrocytes or committed neuronal progenitors (OPC: Oligodendrocyte Progenitor Cells; INP: Intermediate Neuronal Progenitors) are, respectively, in blue, purple and blue-green colors; gene lists enriched in the putative GSC and DGC populations are highlighted in orange and in light blue, respectively. Signatures are available in Supplementary Table 1. GSEA calculated FDR adjusting for multiple comparisons; details of p-value and FDR calculation are described in the GSEA website (http://software.broadinstitute.org/gsea/index.jsp). Related to Fig. 1c. c, Log2 expression levels of the indicated oRG (top graphs), NSC and GSC (middle graphs) and INP markers (bottom graphs) in the subpopulations of neoplastic cells of the Darmanis dataset that are at the start (GSC, n = 221 cells) and at the end (DGC, n = 221 cells) of the pseudotime trajectory depicted in Fig. 1c. Data are presented as mean + s.d. p-values were determined by unpaired two-tailed t test. d, RNA velocities (arrows) of neoplastic cells of the Darmanis dataset projected in the space of the first two principal components. Red and blue dots are the cells that are at the start (GSC) and at the end (DGC) of the pseudotime trajectory depicted in Fig. 1c.

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