GSM5016858: 3MO_MG_C9_KO_HOMO_1; Mus musculus; RNA-Seq
The meninges were removed from the mice brains of different genotypes and dissociated using the Neural Tissue dissociation kit, papain based (Miltenyi Biotech) and GentleMACSdissociator (NTDK Brain setting). The lysate was collected and passed through a 70μm strainer to obtain a single cell suspension. Next, myelin was removed from the cells by incubating with magnetic myelin beads (Myelin Removal Beads II, human, mouse, rat, Miltenyi Biotech) and removed using the AutoMACs. Cells were then incubated with CD11b+ magnetic beads CD11b MicroBeads, human and mouse, (Miltenyi Biotech) and sorted using the AutoMACs. After sorting, cells were pelleted and stored at -80°C until further downstream application.  RNA was isolated using Qiagen RNeasy Micro Kit (Cat #74004) according to manufacturer's instructions. RNA-seq libraries were generated using 75ng to 180ng total RNA as input for the TruSeq Stranded mRNA Library Prep kit (Illumina) according to the manufacturer's protocol, and samples were indexed using TruSeq RNA Single Indexes (Illumina). Library preps were analyzed via bioanalyzer (Agilent) and quantified via Qubit fluorometric quantification (Thermo Fisher). Quantified libraries were normalized, pooled, and sequenced on a NextSeq 500 sequencer (Illumina) using the single-end 75 nucleotide setting