GSM4223663: F-12: CON rep1; Mus musculus; RNA-Seq
Single nucleus dissociation was completed with a modified protocol based on our previous work64. Briefly, animals were euthanized by isoflurane inhalation and cervical dislocation. The thoracic SCI site was rapidly dissected and frozen on dry ice. Spinal cords were dounced in 500 µl sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg-acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 with the Kontes Dounce Tissue Grinder. 2 mL of sucrose buffer was added and filtered through a µm cell strainer. The lysate was subsequently centrifuged at 3200 g for 10 min at 4°C. The supernatant was decanted, and 3 mL of sucrose buffer added to the pellet and incubated for 1 min. The pellet was homogenized using an Ultra-Turrax and 12.5 mL of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg-acetate, 1 mM DTT) was added below the nuclei layer. The tube was centrifuged at 3200 g at 4°C and supernatant immediately poured off. The nuclei on the bottom half of the tube wall were collected with 100 µl PBS with 0.04% BSA and 0.2 U/µl RNase inhibitor. Resuspended nuclei were filtered through a 30 µm strainer. The nuclei suspension was finally adjusted to 1000 nuclei/µl.  Library preparation was carried out with 10X Genomics Chromium Single Cell Kit Version 2. The nuclei suspension was added to the Chromium RT mix to achieve loading numbers of 5000. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer's instructions were followed. 