In parallel, the Gitler and Le Pichon labs performed similar sets of experiments with independent methods, interpetation, and validation. Still, the conclusions from these experiments are remarkably similar, underscoring the fundamental biology uncovered in their collective works. Both datasets are integrated on this site and provided for download under the 'More Information' tab. They are independently marked by 'Gitler Lab' and 'Le Pichon' Lab metadata in the downloadable h5 matrices as well as the only analysis portal.
Four to 8 adult mice (P100-P150) in five independent experiments were euthanized with CO2 and decapitated caudal to the brain stem. Their spinal columns were severed just caudal to the sacral spinal cord, and rapidly cut out as described. Briefly, a blunt 18-gauge syringe containing ice-cold PBS was inserted into the caudal end of the spinal cord and used for rapid hydraulic extrusion of the entire, intact cord. For droplet-based snRNA-seq, libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v.3 according to the manufacturer’s protocol (10x Genomics). The generated snRNA-seq libraries were sequenced using NextSeq 500/550 High Output v2 kits (150 cycles) to an average read depth of ~500,000 reads/cell. Most subsequent analysis was performed using the Seurat R Package. All 43,890 transcriptomes were normalized for read-depth with the CellRanger aggr function, and then loaded into Seurat. The nuclei were batch-corrected using the Seurat Integrate function as previously described2, which enables cells from different experiments to be projected into the same high and low-dimensional spaces. Principal component analysis (PCA) was performed on the whole dataset, and the top 15 components were used to generate a uniform manifold approximation (UMAP).