GSM8634129: Naïve_scRNAseq; Rattus norvegicus; RNA-Seq
Rats were euthanized with 1 mL of Euthanyl injected intraperitoneally, and then transcardially perfused with Hanks' Balanced Salt Solution (HBSS, ThermoFisher, 14175-103). All spinal cords were dissected and a 2 cm section surrounding the lesion site was isolated within 90 min after perfusion and maintained on ice for the duration of the cell sorting experiment. Spinal cords were diced in HBSS and then collected and placed into Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11960069) and deoxyribonuclease (5 μg/mL, Worthington, L3002007). Collected tissue was mechanically dissociated and dounced 5 times with a loose-fitted dounce homogenizer; following which the suspension was passed through 70 μm and 40 μm filters. Cells were then pelleted, resuspended in 5.4 mL of HBSS and myelin debris was removed using 1800 μl of Debris Removal Solution (Miltenyl Biotec, 130-109-398) and centrifugation at 3000 G for 10 min. The myelin debris was removed and cells were topped up to 15 mL with HBSS and centrifuged at 1000 G for 10 min. Cells were passed through a 20 μm filter, following which cells were resuspended in Fc block Anti-Mo CD16/CD32 (1:200, Invitrogen, 14-0161-85) blocking solution for 5 min, and subsequently stained with ZombieAqua (1:50, Biolegend, 423101), DRAQ5 (1:1000, ThermoFisher, 62240) and Cd11b (1:50, BD BioSciences, 561684) for 20 min. Cells were resuspended in a solution of 1% bovine serum albumin (BSA; Sigma-Aldrich, A8806) dissolved in HBSS and then were isolated using fluorescence-activated cell sorting (FACS) on a Sony MA700 and gated for live, nucleated and Cd11b+ cells. Following sorting, cells were manually counted using a hemocytometer, pelleted and resuspended in HBSS. The FACS-isolated cells were processed using the 10x Genomics Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics; 1000121) and sequenced on the Illumina HiSeq P150 Sequencer (Novogene). The samples were paired-end, single index sequenced to an average of 40,000 reads per cell.