title: GSM8601551: CNS-Myeloid Vehicle Rep3; Mus musculus; RNA-Seq
library_construction_protocol: On day 35 mice were euthanized by isoflurane overdose and perfused with chilled PBS containing 5μg/mL actinomycin and 10μM triptolide. Brains and spinal cord were extracted and mechanically dissociated through a 16G needle five times in PBS with actinomycin and triptolide. Dissociated samples were spun 2 minutes at 200g and resuspended in papain digestion buffer with actinomycin and triptolide. Samples were enzymatically dissociated in papain for 10 minutes at 37c with slow rotation then triturated and passed over a 100μM filter. Samples were spun for 10 minutes then resuspended in PBS and Miltenyi Debris Removal Solution to remove myelin following manufacturer's protocol. Samples were then incubated with TruStain Fc Blocker (1:200) and Live/Dead Aqua (1:2000) for 20 minutes at RT. Cells were washed and stained in 0.5% BSA, 1mM EDTA with Ly6G eFluor 450 (1:250), CD11b PerCP-Cy5.5 (1:250), CD45 APCFire750 (1:250), and stained for 30 minutes at RT, then washed in 1mL and resuspended in 200μL of 0.5% BSA, 1mM EDTA and filtered. Cells were then sorted on Viable,CD45+,CD11b+,Ly6G- into RLT lysis buffer. RNA was then isolated using Qiagen RNEasy Plus Mini Kit Unstranded poly-A mRNA Seq libraries were prepared from RNA isolated from sorted cells