title: GSM5260020: CD11b+ MG-KO-rep1; Mus musculus; RNA-Seq
library_construction_protocol: At P6, toe clips were obtained to determine genotypes. At P8, the mice were anesthetized with ketamine and xylazine, and transcardially perfused with 10 ml PBS.  The brains of pups with the same genotype were pooled and digested in 5 ml Stempro Accutase (A11105-01; Life Technologies, Carlsbad, CA) at 37°C for 30 min. Every 5 min during the incubation, the tube was inversed several times. After the 30-min incubation, the enzyme activity of Accutase was blocked by addition of 2 ml FBS. The digested tissue was then filtered through a 70-μm cell strainer to make a single cell suspension. Cells were centrifuged and resuspended in 5 ml 35% Percoll (P4937; Sigma-Aldrich) in PBS. 3 ml of 70% Percoll was slowly added to the bottom using a Pasteur pipette. The tubes were then centrifuged at 2,000 × g for 20 min at 18°C with the brake set to 0. The top lipid/myelin layer was quickly removed using a pipette. Cells in the interphase between the 35% and 70% Percoll layers were harvested with a Pasteur pipette, washed by centrifugation and resuspended in ice-cold PBS. The cells were stained with α-ms CD11b APC (#17-0112; eBioscience), washed, and resuspended in PBS containing propidium iodide and 1 mM EDTA. CD11b+PI- cells were FACS-sorted and resuspended in 500 µl Tri Reagent, flash frozen, and stored at -80°C until use. RNA was extracted with the Directzol RNA isolating kit (Zymed).  Samples with high RNA integrity number (>8) were used for library construction. RNA libraries were prepared for sequencing using standard Illumina protocols. Each sample (200 ng) was poly-A selected, fragmented, and library prepared using the TruSeqRNASample Prep Kit (Illumina).