GSM5333257: WT normal_8; Mus musculus; RNA-Seq
Four days post-injury animals were euthanized and perfused with ice cold GKN buffer (8 g/L NaCl, 0.4 g/L KCl, 1.41 g/L Na2HPO4, 0.6 g/L NaH2PO4, and 2 g/L D(+) glucose, pH 7.4) through the heart to eliminate circulating blood.  Brains were harvested and ipsilateral hemispheres were dissected for further study.  Ipsilateral brain hemispheres were harvested and the olfactory bulbs removed. Tissues were minced and washed in cold GKN buffer.  Tissue chunks were resuspended in 2.5 mL of digestion buffer (NOSE buffer (4 g/L MgCl2, 2.55 g/L CaCl2, 3.73 g/L KCl, 8.95 g/L NaCL, pH 6-7) with 200 U/ml DNase I (Sigma-Aldrich) and 0.2 mg/mL Collagenase I (Worthington Biochemical)) and incubated at 37C in a dry incubator for 1h with shaking every 15 min. All samples were placed on ice to halt enzymatic activity.  Tissues were crushed through a 100 micron nylon filter cup (BD Biosciences) and the cell suspension was washed with GKN buffer. An isotonic Percoll solution (90% Percoll (GE Biosciences), 10% 1.5M NaCl) was made and brought to room temperature.  Cells were resuspended in 20 mL of a 1.03 g/mL isotonic Percoll solution in GKN buffer and underlayed with 10 mL of a 1.095 g/L isotonic Percoll solution in PBS.  Cells were spun at ~850g at room temperature for 20 min. Buffy layers were isolated.    Cells were blocked with 10% rat serum for 10 min on ice and then stained with the following antibodies: anti-CD45 PE-Cy5.5 (Clone 30-F11, Invitrogen), anti-Ly6G PE-eFluor610 (Clone 1A8, Invitrogen), anti-CD11b PE (Clone M1/70, BD Biosciences), anti-Ly6C PE-Cy7 (Clone AL-21, BD Biosciences), and anti-CD3 FITC (Clone 17A2, BD Biosciences).  DAPI (Invitrogen) was used at 1 uM to gate out dead cells.   Cells were sorted on a FACS AriaIIu (BD Biosciences) at the San Francisco VA Medical Center Flow Cytometry Core Facility. Single cell RNA seq was performed at the Genomics Core Facility at the Institute for Human Genetics (University of California, San Francisco) using the 10X Genomics platform with gel emulsion bead technology.   Chromium Single Cell 3' Reagent Kits v3 were used according to manufacturer protocols and each sample was run on separate lanes.   Libraries were sequenced on a NovaSeq6000 at the UCSF Center for Advanced Technologies with an average of 2967 average UMI per cell.