title: GSM8059226: CD45- EAE Brain, C3 negative; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized with isoflurane followed by perfusion with chilled HBSS. Brains were extracted, chopped into small pieces, then digested with 20U/mL Papain and 100U/mL DNAse I in digestion buffer (EBSS with 22.5mM D(+)-glucose, 26mM NaHCO3, 0.5mM EDTA, and 1mM L-cysteine at pH 7.4) under slow rotation at 37c  for 45 minutes with manual trituration steps every 15 minutes. After filtering, myelin debris was removed with 30% percoll solution. Cells from 7 animals (3 male, 4 female) were pooled and stained for viability (L/D Fixable NIR) and CD45 (anti-CD45 Pacific Blue) then two populations were sorted: viable;CD45-;C3-tdTomato+ and CD45-;C3-tdTomato-. Following sorting, cells were washed, counted, and loaded onto 2 seperate lanes (Pos cells in 1 lane, Neg cells in the other) of a 10X Chromium Chip G then partitioned into GEMs on a Chromium Controller following manufacturer's recommended protocol. Libraries were constructed and sequenced using 10X 3' 3.1 chemistry according to manufacturer's recommendations