Identification of astrocyte regulators by nucleic acid cytometry.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) 1 . Astrocytes are heterogeneous CNS-resident glial cells that participate in the pathogenesis of MS and its model experimental autoimmune encephalomyelitis (EAE) 2,3 . However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. To address these challenges, we developed FIND-seq (Focused Interrogation of cells by Nucleic acid Detection and Sequencing), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids, and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in MS and EAE 4 . Using FIND-seq in combination with conditional knock-out mice, in vivo CRISPR/Cas9-driven genetic perturbation studies, and bulk and single-cell RNA-seq analyses of mouse EAE and human MS samples, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, FIND-seq enabled the identification of a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq allows the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.