GSM6216168: Mouse-SC-RNAseq-Middle-aged-naïve; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane and perfused with 1x PBS. The spinal cord was flushed through the spinal canal with PBS. Spinal cord tissues were homogenized with an 18-gauge needle in a protease inhibitor solution created using protease inhibitor cocktail tablets (Roche) per the manufacturer's protocol, centrifuged at 800 g for 5 minutes, and the supernatant was collected and stored at -80°C. Tissue pellet was resuspended in a solution of HBSS with 1 mg/ml collagenase A (Roche) and 1 mg/ml DNase I (Sigma-Aldrich) and incubated at 37°C for 20 minutes. Mononuclear cells were separated from myelin via centrifugation in a 27% Percoll solution (GE Healthcare). Cells were stained for CD45 and sorted via flow cytometry. Mononuclear cells were isolated from 4 spinal cords per sample. Libraries were sequenced on an Illumina Novaseq instrument and counted with CellRanger v3.1.0 using mm10 reference genome GENCODE vM23/Ensembl 98.