Brains were rapidly dissected, cooled in ice-cold Hibernate-A for 1.5 minutes, transferred to PBS, and prepared under a dissecting microscope. The cortex and cerebellum were removed to expose the superior colliculus (SC). The sSC was visualized coronally, micro-dissected by knife cuts along its visually approximated anatomical borders, and processed into a single-nucleus suspension using a published detergent-mechanical lysis protocol (Matson et al., 2018). Briefly, the sSC was homogenized in a lysis buffer, and cell nuclei were purified by density gradient centrifugation. After resuspension, nuclei were labeled with propidium iodide (Propidium Iodide Ready Flow™ Reagent, Invitrogen, catalog # R37169) and then counted with a CellDrop automated cell counter (DeNovix, Delaware, USA). We used the 10X Genomics Chromium Next GEM 3' kit v3.1 according to the manufacturer's protocol (user guide revision D) to encapsulate each nucleus's poly-adenylated RNA and then process the poly-adenylated RNA into cDNA sequencing libraries. We then sequenced the cDNA libraries by Illumina Next-Seq 2000 to an average of 29,857 mean reads per cell.