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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: CLEAR: coverage-based limiting-cell experiment analysis for RNA-seq

Fig. 4

Application of CLEAR to a mouse neural lcRNA-seq experiment. a Schematic of cell isolation and preparation for sequencing. The dentate gyri of Nestin-GFP mice were microdissected and dissociated into a single cell suspension. Cells were labeled with fluorescently conjugated antibodies against markers for specific populations of cells present in the hippocampus. GFP+ GLAST+ stem cells, GFP+ GLAST− progenitor cells and GFP-GLAST+ astrocytes were isolated from live cells that were negative for microglial, oligodendroglial and endothelial markers. SMART-seq libraries were generated from these sorted cells; b The means and ranges of CLEAR transcripts from each cell type (4 biological replicates per group). All groups are significantly different when compared using a t test (p < 0.01); c PCA analyses by murine neuronal cell types. Top Panels: PCA plots using all available transcripts; Bottom Panels: PCA plots using only CLEAR transcripts; d Normalized DESeq2 transcript counts for 4 genes that pass CLEAR and are known to be differentially expressed in murine neural stem cells, progenitors and astrocytes are used to confirm the identity of the cell populations derived from the staining and FACS strategies used to enrich these three cell populations. Boxplots: orange line, mean CLEAR transcripts for four biological replicates per neural cell type; whiskers: displaying 1.5× the inter-quartile range (IQR) beyond the first and the third quartiles; circles: outliers. FACS fluorescence activated cell sorting, PC principal component

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