GSM9038450: snRNA-seq_OpossumV1-2; Monodelphis domestica; RNA-Seq
Opossums were deeply anesthetized with isoflurane followed by decapitation, and the brain was immediately removed and submerged in ice-cold Hibernate A (BrainBits Cat. # HACA). Anatomical landmarks (Wong and Kaas, 2009) were used to identify area 17, the primary visual cortex (V1). Extracted brains were placed in an ice-cold metal mold (Kent Scientific Cat. # RBMS-300C) and a 2 mm coronal slice was obtained by bisecting the superior colliculus with a razor blade (Personna Super) and inserting a second blade 2 mm anterior to the first. This slice was immediately placed in Hibernate A and cuts were made lateral to V1 (~3 mm from the midline) in both hemispheres. V1 (from both hemispheres) was then removed from the underlying white matter and immediately transferred to a cryogenic vial (Millipore Sigma Cat. # CLS431417) and flash frozen in liquid nitrogen. Tissue was stored in liquid nitrogen for ~1 year before single-nucleus RNA-sequencing was performed. Nuclei were isolated from each biological replicate using the 10x Genomics Chromium Nuclei Isolation Kit. Both biological replicates were processed in parallel by two different experimenters. To remove excess debris (previously observed in adult opossum nuclei preparations), we performed fluorescence-aided nuclear sorting (FANS) for Draq5-positive nuclei. After FANS, nuclei suspensions were counted on a hemocytometer using an ethidium bromide counterstain. Nuclei suspensions were examined for blebbing under a brightfield microscope before dilution to 700-1200 cells per µL. Nuclei suspensions from each biological replicate were loaded into two wells of a 10x Chromium 3' v3 chip, each targeting 10,000 cells. Droplet RNA sequencing experiments using the 10x Chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina NovaSeq™ X Plus Sequencing System to a depth of ~30,000 reads per cell.