title: GSM9500021: Mouse lumbar spinal cord snRNA-seq_experiment1; Mus musculus; RNA-Seq
library_construction_protocol: 8-week old male and female mice were anesthetized with isoflurane and decapitated. Vertebral column was rapidly dissected out and transferred to ice-cold carbogenated (95% O2 and 5% CO2) NMDG-HEPES-ACSF (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 10 mM MgSO4, 1 mM CaCl2, 1 mM kynurenic-acid Na salt, 5 mM Na-ascorbate, 2 mM thiourea and 3 mM Na-pyruvate, pH adjusted to 7.4, osmolarity ranging 300–310 mOsm). L1-L5 lumbar spinal cords were microdissected out and cut into small 1 mm2 size pieces. Microdissected spinal cord pieces were transferred to a Kimble Dounce tissue-grinder containing 750 microL of Lysis Buffer (0.1% Triton-X100, 320 mM sucrose, 10 mM Tris-HCl – pH=7.4, 3mM MgCl2, 10 mM NaCl, 0.5% RNAse free BSA, 1% Kollidon VA64, 1mM dithiotreitol, 0.1 U/microL Roche Protector RNAse inhibitor in DNAse/RNAse free ddH2O). Tissue was homogenized with 5 strokes with loose pestle and 15 strokes with tight pestle and resulting nuclear suspension was strained with a 40 micron cell strainer and washed with 1.25 mL of Nuclear Buffer (320 mM sucrose, 10 mM Tris-HCl – pH=7.4, 3 mM MgCl2, 10 mM NaCl, 0.5% RNAse free BSA, 1% Kollidon VA64, 1mM dithiotreitol, 0.1 U/microL Roche Protector RNAse inhibitor in DNAse/RNAse free ddH2O). Cell suspension is layered onto 12 mL of 1M sucrose cushion (1M sucrose, 10 mM pH=7.4, Tris-HCl (pH=7.4), 3 mM MgCl2, 10 mM NaCl, 0.5% RNAse free BSA, 1% Kollidon VA64, 1mM dithiotreitol, 0.1 U/microL Roche Protector RNAse inhibitor in DNAse/RNAse free ddH2O) in a 5 mL Oakridge tube. The latter are centrifuged at 500g for 5 min at 4C in a spin-out-rotor. The sucrose cushion is decanted and pure nuclei are resuspended in 300 µL of nuclear buffer containing 10 µg/mL 7-AAD and 1:100 custom conjugated anti-NeuN-Alexa647 antibody (Biolegend #608452). The nuclei are incubated for 15 min at 4C. Thereafter, nuclei are spun down at 500g for 5 minutes and washed twice with 3 mL of nucleus buffer and resuspended in 1 mL of nucleus buffer. The resulting nucleus suspension is FACS sorted to enrich for NeuN/7-AAD double positive cells (neurons). The neuronal nuclei are concentrated with CUTANA ConA beads per manufacturer's protocol and resuspended in 1x Nucleus Buffer from 10x Genomics with resulting nuclear suspension loaded to the 10x Genomics microfluidic chip and processed per manufacturer's protocol. Single-nucleus suspensions were processed on a 10X Chromium Xi instrument using the Chromium Next GEM Single Cell Multiome Reagent Bundle (#100028) following the manufacturers protocol. The resulting sequencing libraries were sequenced on a Novaseq X platform (PE 150 bp). Libraries were sequenced to a minimum depth of 50,000 reads per nucleus.