GSM9201845: AR-97Q_8w_spinal cord; Mus musculus; RNA-Seq
Mice were deeply anesthetized, and the entire spinal cord were dissected and snap-frozen in powdered CO2 in liquid nitrogen. Spinal cords from 4 mice for each condition were pooled for homogenization and nuclear isolation. Fresh frozen murine spinal cord was placed in a Dounce homogenizer (Kimble Chase 2 ml Tissue Grinder) containing ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal and 0.2 U/μl RNase inhibitor). After homogenizing, the lysate was diluted with 1.4 mL of lysis buffer and incubated for 5 minutes. The homogenate was passed over a 70 μm strainer (Fisher Scientific). The filtered lysate was centrifuged at 500 × g for 5 min at 4 °C. After centrifugation, the pellet was resuspended in 1.5 ml lysis buffer and incubated for 3 minutes on ice. Then, the pellet was centrifuged at 500 × g for 5 minutes and resuspended in 1500 µl of wash buffer (1× PBS with 1% BSA and RNase Inhibitor). The nuclei were washed and centrifuged at 500 × g for 5 minutes three times and filtered through a 40 µm cell strainer. The cell suspensions were processed according to the debris removal solution (Milteni Biotec) protocol. Cells were resuspended in cold PBS with 0.04% BSA and stained with DAPI. The nuclei were sorted with a FACS Aria instrument (Becton Dickson) with a 100 nm nozzle and 405 nm excitation laser. These nuclei were centrifuged, inspected for appearance, and concentrated for the next step. The nulei per sample were run on the Chromium GEM-X Single Cell Gene Expression 3' v4 platform (10x Genomics, Pleasanton, CA). DAPI-positive nuclei were immediately loaded onto a Chromium Single Cell Processor (10x Genomics) to barcode the mRNA inside each nucleus. Sequencing libraries were constructed according to the manufacturer's instructions, and the resulting cDNA samples were run on an Agilent Bioanalyzer using the High Sensitivity DNA Chip as a quality control to determine cDNA concentrations. The samples were run on NovaSeqX Ten with 150 bp paired end to obtain ≥20,000 reads per cell. A total of 22,093 and 18,372 cells with approximately 423 million and 541 million reads were sequenced for the AR-97Q and AR-24Q samples, respectively. Reads were aligned and assigned to Ensembl mm10 transcript definitions using the Cell Ranger v9.0.1 pipeline (10x Genomics). The gene barcode matrices for each sample were imported into R (version 4.4.3) using the Read10X function in the Seurat R package (version 5.3.0).