GSM9598434: VNIM non-lesion white matter; Mus musculus; RNA-Seq
Neutral red–labeled white matter was isolated as lesion tissue. Non-lesion tissue consisted of approximately 1 cm of white matter collected 0.5 mm above and below the lesion site. In naïve animals, where no neutral red labeling was observed, approximately 2 cm of white matter from a comparable spinal cord level was collected. To obtain sufficient cell numbers, lesion and non-lesion samples were pooled from two animals of the same sex. Dissected tissue was transferred to gentleMACS C Tubes (Miltenyi Biotec) containing digestion buffer from the Adult Brain Dissociation Kit (Miltenyi Biotec) supplemented with the inhibitor cocktail and mechanically minced. Enzymatic dissociation was performed using the gentleMACS OctoDissociator (program 37C_ABDK_01). Samples were briefly centrifuged, filtered through a 70 μm strainer, washed, and subjected to debris removal by density gradient centrifugation according to the manufacturer's instructions. Cell viability and concentration were assessed using ViaStain AOPI staining solution (1:1) and quantified using a DeNovix CellDrop FL prior to library preparation. To minimize ex vivo transcriptional and translational activation, an inhibitor cocktail containing actinomycin D (5 μg/mL; Sigma-Aldrich), triptolide (10 μM; Sigma-Aldrich), and anisomycin (27.1 μg/mL; Sigma-Aldrich) in 1x PBS without Ca2+, Mg2+, or phenol red (pH 7.4; Gibco) was applied during tissue dissection and enzymatic digestion. Single-cell libraries were generated using the GEM-X Universal 3′ Gene Expression v4 4-plex OCM Reagent Kits (10x Genomics) according to the manufacturer's protocol, targeting 5,000 cells per sample. Quality control for cDNA and final libraries was performed using the Agilent 2100 Bioanalyzer system using the Agilent High Sensitivity DNA quantification kit (Agilent Technologies). Libraries were sequenced by MedGenome on an Illumina NovaSeq platform using 10x Genomics-recommended read configurations, targeting 40,000–80,000 reads per cell.