Gene Expression
Nebulas
Gene Expression NebulasSummary: Intra-tumor genetic heterogeneity comes from whole chromosome and/or focal copy number variations (CNVs). We investigated the impact of whole chromosome CNVs on gene expression by performing single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line. From the gene expression data, we computationally inferred large-scale CNVs in single cells. We find that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we find that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival. In conclusion, this study demonstrates the utility of single-cell RNA sequencing when analyzing chromosomally unstable cells.
Overall Design: Single-cell RNA sequencing of normal neural stem cells and glioblastoma cancer stem cells using C1 Single Cell Auto Prep system from Fluidigm and Illumina Nextera XT library sequencing as C1- protocol.
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| Growth Protocol: | Cells were grown in serum-free culture conditions as described in Pollard et al, Cell Stem Cell, 568-580, 2009. |
| Treatment Protocol: | The cells were dissociated, filter strain with 40m, resuspended in the serum free culture medium. |
| Extract Protocol: | Single cells were caputured, lysed, and cDNA prepared using the C1 Single Cell Auto Prep system from Fluidigm. |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |