GSM9455128: Sample 1: Mouse Lateral Septum Cells for snRNAseq (S4); Mus musculus; RNA-Seq
Tissue collection, preparation, sorting, and sequencing procedures were performed following previously published protocols. 8 naïve, adult (3 months old) male C57BL/6J littermates were used for tissue collection. Mice were allowed to acclimate in the lab in their homecages for ≥1 hr prior to being deeply anesthetized as above and perfused using an ice-cold (4 ºC) HEPES-sucrose solution (pH=7.4; in mM): 110 NaCl, 10 HEPES, 25 glucose, 75 sucrose, and 7.5 MgCl2·6H2O. Brain tissue was then immediately dissected and mounted on a vibratome, and 200 µm-thick coronal sections (spanning the broad anterior-posterior/dorsal-ventral extent of the LS) were collected and maintained in the HEPES-sucrose solution. 500 µm-wide circular punches across the anterior-posterior and dorsal-ventral axes of the LS from each mouse were made, and immediately flash frozen using liquid nitrogen. Care was taken to minimize the collection of any non-LS tissue; however, we recognize the possibility for the partial inclusion of some neighboring structures such as medial septum, triangular septum, prefrontal cortex, and/or striatum, given their proximity to the LS. Frozen septal tissues were immediately collected into Eppendorf tubes and maintained at -80 ºC until sequencing. Sample preparation and nuclei isolation from the samples was performed using fluorescence-activated nuclei sorting (FANS) following previously published protocols. snRNAseq was then performed using the 10X Genomics (v.3) kit and using NextSeq or NovaSeq4000 (Illumina), following manufacturer's protocols and instructions (three libraries were generated from pooled cells).