A reliable work ow for single-nuclei RNA-seq of hippocampal adult-born granule cells

ARIEL BERARDINO; LUCIANA FERELLA; ARIEL CHERNOMORETZ; M. LUZ VERCESI; JULIANA BROWN; ALEJANDRO F. SCHINDER; NATALÍ B. RASETTO; TOMÁS VEGA WAICHMAN; PAOLA ARLOTTA; DAMIANA GIACOMINI

Congreso; Congreso XXXV de la Sociedad Argentina de Neurociencias (SAN) con formato virtual; 2020

Adult hippocampal neurogenesis plays a critical role in spatial memory, retrieval, context discrimination, and clearance of memory traces. In the mouse dentate gyrus, adult-born granule cells (GCs) maturation process lasts for more than 8 weeks and can be divided into 4 discrete phases. Although the principal electrophysiological and morphological distinctive features of each stage have already been described in our laboratory, the molecular mechanisms that control the onset and progression towards a fully mature phenotype are still an open question. Our goal is to investigate the molecular mechanisms instructing structural and functional maturation and synaptogenesis in each developmental stage of adult-born GCs. In this work, we set up an efficient protocol for high-throughput single-nuclei RNA-sequencing using 10X Genomics Chromium technology from adult-born GCs. We use double transgenic mice, Ascl1CreERT2;CAGfloxStopSun1GFP, to allow conditional expression of Sun-1/sfGFP in the inner nuclear membrane of adult-born GC upon tamoxifen induction. At specific time points, dentate gyri were microdissected, and fluorescent nuclei were isolated and purified using FACS. Finally, we present preliminary results of the transcriptomic profile analysis proceeding from the experimental 2-weeks point. Therefore, the presented single-nuclei RNA sequencing workflow represents a reliable method to explore the molecular principles of neuronal maturation and integration in the adult brain.