Single nucleus RNA-seq of cell diversity in the adult mouse hippocampus.

Habib N, Li Y, Heidenreich M, Swiech L, Avraham-Davidi I, Trombetta J, Hession C, Zhang F, Regev A. Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.Science  28 Jul 2016 DOI: 10.1126/science.aad7038
Contact: naomi@broadinstitute.org

Single cell RNA-Seq provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by EdU to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. This study contains the sNuc-Seq analysis performed as a part of the Div-Seq method development.

Using sNuc-Seq, we analyzed 1,367 single nuclei from hippocampal anatomical sub-regions (DG, CA1, CA2, and CA3) from adult mice, including enrichment of genetically-tagged lowly abundant GABAergic neurons (9). sNuc-Seq robustly generated high quality data across animal age groups (including 2 years old mice), detecting 5,100 expressed genes per nucleus on average, with comparable complexity to single neuron RNA-Seq from young mice (1, 2, 3). Analysis of sNuc-Seq data revealed distinct nuclei clusters (Fig. 1B-D shown below) corresponding to known cell types and anatomical distinctions in the hippocampus.

Fig. 1 sNuc-Seq identifies cell types in adult mouse brain.(A) Representative images of isolated nuclei are more uniform than of dissociated neurons from adult brain. Scale = 10μm. sNuc-Seq method (right): nuclei are isolated, FACS sorted and profiled using modified Smart-SEq. 2 (21). (B) Major cell types identified from sNuc-Seq data reflected by clusters, shown as 2-D embedding of 1,188 nuclei from adult mouse hippocampus. (C) Cluster-specific genes across single nuclei. Color bar matches cluster color in B. (D) Identification of DG granule cell, CA1, CA2, and CA3 pyramidal cell clusters, by marker genes, shown as: 1, ISH image in hippocampus section (10) (arrowhead: high expression; Scale = 400μm.); 2, histogram quantifying expression in relevant cluster; and 3, 2-D embedding of nuclei (as in B) colored by relative expression.