Single cell RNA-Sequencing of adult NSCs and related populations from the sub-ventricular zone. Mus musculus strain:FVB/N-Tg(GFAPGFP)14Mes/J

To define the molecular heterogeneity of four adult cell populations in the SVZ (astrocytes, quiescent and activated NSCs, and more committed NPCs), we performed single cell RNA-sequencing for these four putative populations. We implemented a well-accepted FACS protocol to freshly isolate adult populations from the SVZ. Briefly, as described in (Codega et al., 2014), SVZs were microdissected from the lateral ventricular walls of young male mice (3 months old) that express green fluorescent protein (GFP) under the GFAP promoter. Single cells were dissociated from the tissue and were stained with markers of NSC identity and activation, including CD133/Prominin 1 [PROM1] and EGFR. Cells were all negatively selected for markers of neuroblasts and ependymal cells (CD24), endothelial cells (CD31), and immune cells (CD45). This approach enabled us to isolate niche astrocytes (GFAP-GFP+ PROM1- EGFR-), qNSCs (GFAP-GFP+ PROM1+ EGFR-), aNSCs (GFAP-GFP+ PROM1+ EGFR+), and NPCs (GFAP-GFP-EGFR+), as described in (Codega et al., 2014) (Figure 1A, Figure S1A). Each of these enriched populations was used to prepare single cell RNA-sequencing libraries using the Fluidigm C1 Single-Cell Auto Prep microfluidic system, using the small sized chip (Wu et al., 2014). Cells were subjected to live/dead assessment on the chip, and dead cells were excluded from the analysis. Pooled and barcoded single cell libraries were sequenced on Illumina MiSeq, and a subset of libraries was later sequenced on Illumina HiSeq 2000 to achieve greater sequencing depth (Table S1).