Div-Seq – A single nucleus RNA-Seq method reveals dynamics of rare adult newborn neurons in the CNS

Transcriptomes of individual neurons provide rich information about cell types and dynamic states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation from dense adult tissue is challenging, and markers for each phase are limited.

Researchers from the Broad Institute have developed Div-Seq, which combines Nuc-Seq, a scalable single nucleus RNA-Seq method, with EdU-mediated labeling of proliferating cells. They first show that Nuc-Seq can sensitively identify closely related cell types within the adult hippocampus. They apply Div-Seq to track transcriptional dynamics of newborn neurons in an adult neurogenic region in the hippocampus. Finally, the researchers find rare adult newborn GABAergic neurons in the spinal cord, a non-canonical neurogenic region. Taken together, Nuc-Seq and Div-Seq open the way for unbiased analysis of any complex tissue.

Transcriptional dynamics of adult neurogenesis revealed by Div-Seq

rna-seq

(A) Schematics of Div-Seq method. EdU is injected to mice and incorporates into the DNA of dividing cells (Moore et al., 2015). After isolation, EdU labeled nuclei are fluorescently tagged and captured by FACS for single nuclei RNA-Seq.

(B) Schematics of adult neurogenesis in the dentate gyrus(Ming and Song, 2011). Timing of EdU labeling (tan box) and nuclei isolation are marked.

(C) Div-Seq captured cells expressing known markers of neuronal precursors, neuroblasts and immature neurons. Box plots for the 2 days (2d) and 14 days (14d) EdU labeled nuclei (excluding nuclei classified as non-neuronal). Boxplots shown as in Figure 3G.

(D) Newborn cells form a continuous trajectory. All panels show 2-D embedding of 2d labeled nuclei, 14d labeled nuclei and nuclei from unbiased survey. Nuclei are colored by source (top), by Eomes/Tbr2 marker gene expression (middle), or by Neurod1 marker expression (bottom). Trajectory directionality is chosen by the position of the 2d labeled neurons and known marker genes.

(E) Dynamic gene expression clusters. Four clusters are shown from top to bottom. Left: Running average expression level of the genes in each cluster over the nuclei ordered along the trajectory (as in D). Middle: a heatmap of running average expression of all genes along the trajectory. Red lines mark the transcriptional switches from neuronal precursor cell (NPC) to neuroblast (NB), and from NB to immature neuron. Right: proportions of genes assigned to five major biological pathways

(F) Changes in the composition of the Polycomb Complex (Prc2, top) and the BAF (SWI/SNF) complex (bottom). For each complex, schematics of the complex is shown, and the heatmap of average expression of genes in NPC(NP), NB, immature neurons and mature granule DG cells, and compared to human NPCs (hNPC, absolute log (TPM)).

Habib N, Li Y, Heidenreich M, Swiech L, Trombetta JJ, Zhang F, Regev A. (2016) Div-Seq: A single nucleus RNA-Seq method reveals dynamics of rare adult newborn neurons in the CNS. bioRXiv [Epub ahead of print]. [abstract]

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