GSM8098386: L6_SCI2; Mus musculus; RNA-Seq
For snRNA-seq experiment, single-nucleus dissociation of the mouse lumbar spinal cord was performed according to our established procedures. Following euthanasia by isoflurane inhalation and cervical dislocation, the lower-thoracic and the lumbosacral spinal cord segments were immediately dissected and frozen on dry ice. Spinal cords were doused 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 then added and filtered through a 40 µm cell strainer. The lysate was centrifuged at 3200 g for 10 min at 4°C. The supernatant was then decanted, and 3 mL of sucrose buffer was added to the pellet for 1 min. We homogenized the pellet 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 poured off. 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. Finally, nuclei were resuspended through a 30 µm strainer, and adjusted to 1000 nuclei/µL. snRNA-seq library preparation was carried out using the 10x Genomics Chromium Single Cell Kit Version 3. The nuclei suspension was added to the Chromium RT mix to achieve loading numbers of 2000-5000. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer's instructions were followed.