Conserved genetic signatures parcellate cardinal spinal neuron classes into local and projection subsets [P0NeuronData]

We uncovered orderly genetic tiers that sequentially divide groups of neurons by their motor- sensory, local-long range, and excitatory-inhibitory features. The genetic signatures defining neuronal projections were tied to neuronal birthdate and conserved across cardinal classes. The intersection of cardinal class with projection markers provides a unifying taxonomic solution for systematically identifying distinct functional subsets. In order to minimize technical and biological variabilities, timed-matings were set up to obtain both Vglut2:Cre; tdTomato and Vgat:Cre ;tdTomato neonates at postnatal day 0 on the experiment day. Sex of the animals were visually inspected and verified by genotyping (40). Lumbar segments were dissected from Vglut2; tdTomato (2 female and 1 male) and Vgat; tdTomato (2 female and 1 male) in aCSF. Cell dissociation was conducted using Papain following the manufacturer's instruction (Worthington Biochemical) with 5% Trehalose, 50uM APV, and 800uM KA added during the dissociation process (41). Via FACS (FACSDIVA, BD), 30,000 Vglut2; tdTomato cells and 30,000 Vgat;tdTomato cells were sequentially collected into 500ul of FACS buffer (DMEM w/o Phenol Red; 1mM EDTA; 25mM Hepes pH7.0; 5% FBS). Cell suspension was centrifuged at 300 rcf for 2 minutes, and supernatant was removed leaving 60 ul of FACS buffer and cell pellet. The cells were resuspended, and 33.4 ul of the resuspended cells (~30,000 cells) were loaded onto the 10X Chromium Controller using Chromium Single Cell 3' v2 reagents. Overall design: Vglut2:Cre; tdTomato+ and Vgat:Cre ;tdTomato+ P0 spinal neurons