GSM8129117: Mouse EAE Brain Myeloid C3 Negative; Mus musculus; RNA-Seq
Mice were euthanized with isoflurane followed by perfusion with chilled HBSS containing 10μM triptolide, 27.1μg/mL anisomycin, and 5μg/mL actinomycin D. 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, 1mM L-cysteine,10μM triptolide, 27.1μg/mL anisomycin, and 5μg/mL actinomycin D at pH 7.4) under slow rotation at 37c for 60 minutes with manual trituration steps every 15 minutes. After filtering, myelin debris was removed with debris removal solution (Miltenyi Biotec) following manufacturer's recommended protocol. Cells from 4 animals (3 female, 1 male) were pooled and stained for viability (L/D Violet),CD45 (anti-CD45 APC-Fire750), and CD11b (PerCP-Cy5.5) then two populations were sorted: viable;CD45+;CD11b+;C3-tdTomato- and viable;CD45+;CD11b+;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