GSM6297396: Week 5 - replicate 1; Mus musculus; RNA-Seq
Mice were euthanized with Euthanyl Reg and transcardially perfused with ice-cold HBSS (Invitrogen 14175103). Brains were dissected, processed, and diced in ice-cold HBSS. Collected tissue was then dounced 5 times with a loose fitted dounce and subsequently filtered through 70 and 20μm cell strainers.  Cells were pelleted and myelin debris was removed with Debris Removal Solution (Miltenyi Biotech 130-109-398).  Cells were then blocked for 5 minutes with Anti-Mo CD16/CD32 (Invitrogen 14-0161-85) and stained using Zombie Aqua Fixable Viability Kit (Biolegend 423102), DRAQ5 Fluorescent Probe Solution (Thermofisher 62254) and FITC Mouse anti Rat CD11b (BD Biosciences 561684) for 20 min. After cell staining, cells were sorted on a Sony MA900.  Live nucleated single cells that were CD11b positive were gated and collected.  Final collected cell counts were determined using a hemocytometer. The FACS-isolated cells were processed for single cell sequencing on the 10x Genomics  Chromium Controller (PN-1000121) using the 10x Genomics Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1 (PN-1000121), following the manufacturer's instructions and sequenced on the Illumina HiSeq P150 sequencer (Novogene). We chose the paired end, single indexing form of sequencing with an average read depth of 50,000 reads per cell.