title: GSM8682633: AR97Q_6w_spinal cord; Mus musculus; RNA-Seq
library_construction_protocol: 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 nuclei per sample were run on the 10 × Chromium Single cell 3' gene expression v3.1 platform. Sequencing libraries were constructed according to the manufacturer's instructions, and cDNA samples were run on an Agilent Bioanalyzer using the High Sensitivity DNA Chip as quality control and determination of cDNA concentrations. The samples were run on an Illumina NovaSeq 6000 at Read 1 = 28 bp and Read 2 = 90 bp, with a minimum depth of 20,000 reads per nucleus. For alignment, introns and exons were included in the reference genome (mm10) using the CellRanger v5.0.1 pipeline (10X Genomics). Sequencing data were analyzed using the R package Seurat Version 4.1.3. The gene barcode matrices for each sample were imported into R using the Read10X function.