GSE114000
Identification of region-specific astrocyte subtypes at single cell resolution
Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain-barrier formation and regulation of blood flow, as well as metabolic support of other brain resident cells. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single cell RNA sequencing and generated a database containing ~2000 astrocytes. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. Overall design: Single cell suspension for hippocampus and cortex from P56 C57/Bl6 mice was obtained using a papain-based protocol. A Percoll gradient was used to remove myelin. Astrocytes were then labeled with the ACSA-2 antibody (conjugated to the fluorophore phycoerythrin: PE which specifically recognizes the plasma membrane marker ATP1B2). Cells were isolated by FACS, with one cell deposited per single well of a PCR plate. Preparation of sequencing libraries was done using the optimized Smart-seq2 protocol. Libraries were sequenced using an Illumina NextSeq 500 system (75bp paired-end reads) to optimal coverage (~1 million reads per cell).  Sequencing reads were mapped against mouse reference genome M12 (GRCm38.p5), modified to take ERCC sequences into account using STAR 2.5.2b. Output alignment BAM files were then merged and sorted using Samtools version 1.4. Gene counts were generated using HTSeq version 0.6.1p1. Low quality libraries were removed based on FastQC and additional quality metrics.