GSM7180698: Spinal cord, Injury , P6981_1310; Mus musculus; RNA-Seq
Preparation of single-cell cell suspension and myelin removal was adapted from previously published protocols (Lovatt et al., 2007; Orre et al., 2014). In summary, the procedure was: 5 days after spinal cord crush injury, animals were euthanized by intraperitoneal injection of sodium pentobarbital (200mg/Kg, 100ul i.p., APL) and transcardially perfused with cold Hank's Balanced Salt Solution without calcium or magnesium (HBSS) (Invitrogen). The vertebral column was immediately removed to cold HBSS, the spinal cord was carefully dissected out of the vertebrae and the crush injury segment was separated from the uninjured spinal cord and treated separately hereafter. After removal of the outer meningeal layers, the tissue was dissociated mechanically with a razor blade on a glass petri dish and immediately transferred to cold HBSS. Followed by enzymatic digestion with papain at a final concentration of 8 U/mL with 80 Kunitz units/mL DNase I (Sigma-Aldrich, D4263) in Ca2+/Mg2+-free piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)/cysteine -based buffer, pH 7.4, for 40min at 37°C, 350rpm on a ThermoMixer® (Eppendorf). The tissue was carefully triturated and incubated for additional 5min. The cell suspension was passed through a 70μmCell Strainer (Corning), washed with Minimum Essential Media (MEM) with 1% bovine serum albumin (BSA) and spun down at 200g for 5min at 4°C. The cells were resuspended in MEM and centrifuged over a 90% Percoll gradient (GE Healthcare, 17-0891-01) at 250g for 15min at 4°C. Cells in the lipid layer and below were diluted 5 times in MEM with 1% BSA and spun in a 15mL tube at 250g for 10min at 4°C. All supernatant including the lipid layer were carefully removed and the pellet resuspended in cold MACS Buffer and magnetic Myelin removal beads (Miltenyi biotech, 130-096-433) and incubated for 15min 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 flow through cells were collected, spun down and resuspended in FACS buffer. The single cell suspensions derived from normal spinal cord and injury segments were subject to FACS (fluorescence activated cell sorting) into GLAST-CreER-tomato+/Pdgfrb-eGFP+ double positive and Pdgfrb-eGFP+ single cells, into 96-well plates with Cell lysis buffer (Clonetech, 635013) and frozen to -80°C. Cell sorting was performed on a FACSAria III Cell Sorter system (BD Biosciences). Singlet discrimination was performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H), dead cells were excluded by SYTOX Blue Dead Cell Stain (ThermoFisher Scientific, S34857). Single cells were processed to cDNA libraries according to the Smart-Seq2 protocol  (Picelli et al. 2014). Tn5 tagmentation was performed using the Nextera XT DNA library Preparation Kit (Illumina, FC-131-1002) using dual indexes (i7 and i5).