GSM8243779: Spinal cord, 3d_SCI, 215s_B16; Mus musculus; RNA-Seq
Preparation of single-cell cell suspensions and myelin removal was  performed using adult brain dissociation kit (130-107-677, Miltenyi), followed by Smart-seq 3 library preparation protocol. In summary, uninjured, and 3 days and 5 days after spinal cord crush GLAST-CreERT2;R26R-tdTomato;Pdgfrb-eGFP animals were euthanized by intraperitoneal injection of sodium pentobarbital and transcardially perfused with cold Hank's Balanced Salt Solution without calcium or magnesium (HBSS) (Invitrogen). The spinal cord was carefully dissected out of the vertebrae and the crush injury segment was separated from the uninjured spinal cord tissue and treated separately hereafter. After removal of the outer meningeal layers, the tissue was mechanically dissociated with a razor blade on a glass Petri dish and immediately transferred to cold HBSS. This step was followed by enzymatic digestion. Following dissociation cell pellets were resuspended in cold MACS buffer and magnetic myelin removal beads (Miltenyi Biotech, 130-096-433) and incubated for 15 min at 4°C. The cells were washed and run over MACS MS columns (Miltenyi Biotech, 130-042-201) on a magnetic stand according to the manufacturer's instructions. The cells in the flow through were collected, spun down, resuspended in FACS buffer (2% FBS in PBS) and kept on ice until further processing. The single-cell suspensions generated from GLAST-CreERT2;R26R-tdTomato;Pdgfrb-eGFP animals were subjected to fluorescence activated cell sorting (FACS). Based on reporter gene expression, tdTomato+EGFP+ and tdTomato-EGFP+ cells were collected into 384 plates with Smart-seq 3 lysis buffer mix, followed by a brief centrifugation (1 min, 1500 rpm) and stored at -80 °C until further processing. Cell sorting was performed on a FACSAria III Cell Sorter (BD Biosciences). Singlet discrimination was performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H), and dead cells were excluded by SYTOX Blue Dead Cell Stain (ThermoFisher Scientific, S34857). Single cells were processed to cDNA libraries according to the Smart-Seq3 protocol  (Hagemann-Jensen et al. 2020).