GSM8129111: Male C3 Negative Myeloid, Rep 2; Mus musculus; OTHER
Mice were euthanized with isoflurane followed by perfusion with chilled HBSS containing 27.1μg/mL anisomycin and 5μg/mL actinomycin D. Brains and spinal cords 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 30 minutes with manual trituration steps every 10 minutes. After filtering, myelin was removed with myelin removal beads (Miltenyi Biotec) following manufacturer's recommended protocol. Cells from 2 animals (2 female, 2 male, or 1 male and 1 female) were pooled and stained for viability (L/D Violet),CD45 (anti-CD45 APC-Fire750), CD11b (anti-CD11b BV510), and ACSA2 (anti-ACSA2 APC) then three populations were FACS sorted from each pooled sample: viable;CD45-;ACSA2+;C3-tdTomato+ and C3-tdTomato-, viable;CD45+;CD11b+;C3-tdTomato- and viable;CD45+;CD11b+;C3-tdTomato+. Following sorting, cells were washed and fixed with 4% formaldehyde overnight at 4c, then quenched and stored at -80c. Cells were later thawed and labeled with barcoded probes (10X Genomics Chromium Fixed RNA Profiling) then washed, pooled, and partitioned into GEMs in a single lane of a Chromium Next Gem Chip Q using a Chromium X controller. The single multiplexed library containing 9 samples was generated following manufacturer's (10X Genomics Chromium Fixed RNA Profiling using Multiplexed Samples) guidelines