title: GSM8633414: spinal cord, 2-month-old, PBS, C57/Bl6, sham; Mus musculus; RNA-Seq
library_name: GSM8633414
library_construction_protocol: LPC-lesioned and naïve mice were injected with 0.4 mL 1% Neutral Red31 (Fisher Scientific, Cat. No. N129-25) in 0.1 M phosphate buffer (PB) two hours prior to euthanasia. Mice were euthanized with 0.1 mL Dorminal (Rafter Products, DIN 02333708) and transcardially perfused with 20 mL ice-cold Hanks' Balanced Salt Solution (HBSS; Gibco, Cat. No. 14175-079). The spinal cords were dissected, and the lesions were isolated based visual identification through neutral red staining. Comparable segments of whole spinal cords were isolated as control tissue from naïve mice. Tissue from two mice was pooled per sample, finely micro-dissected on ice in ice-cold HBSS and then transferred to a tube with 1.5 mL Dulbecco's Modified Eagle Medium (DMEM; Gibco, Cat. No. 11960-044) for mechanical dissociation via five oscillations of a loose fitting dounce. The cells were sequentially filtered through 70 µm and 40 µm filters and washed with 7.5 mL DMEM after each filtration step. The cells were pelleted (300 G, 10 min, 4 °C) and resuspended in 6.2 mL HBSS and 1.8 mL Debris Removal Solution (Miltenyi Biotec, Cat. No. 130-109-398). The suspension was mixed by inverting the tube five times and 4 mL of HBSS was carefully overlayed. The suspension was centrifuged (3000 G, 10 min, 4 °C), the top two layers were aspirated and topped up to 15 mL with HBSS. The suspension was mixed by inverting the tube five times and pelleted (1000 G, 10 min, 4 °C).The spinal cords were labelled with Zombie AquaTM Fixable Viability Kit (BioLegend, Cat. No. 423102), DRAQ5TM Fluorescent probe solution (ThermoFisher Scientific, Cat. No. 62254) and FITC anti-mouse F4/80 antibody (Clone BM8; BioLegend, Cat. No. 123107) and sorted on the flow cytometry cell sorter (BD FACSAria III) at the University of Alberta Flow Cytometry Core. The Zombie Aqua-DRAQ5+F4/80+ cells were isolated and cell counts were manually confirmed using a hemocytometer. The samples were processed on the 10x chromium controller following the 10x Genomics Next GEM Single Cell 3' GEM, library, and Gel Bead kit v3.1