GSM8794747: Germ-free, male, rep3; Mus musculus; RNA-Seq
Brains were perfused with PBS then homogenized using a Wheaton dounce tissue grinder (with 0.114 ±0.025mm pestle clearance) in culture media comprised of HBBS with 1.5% HEPES, and 0.5% glucose. Cells were then transferred to a 35% isotonic percoll gradient solution and spun 20 minutes at 700 x g to remove the myelin layer. Transcription (Actinomycin D, Sigma #A1410) and translation (Anisomycin, Sigma #A9789) inhibitors were added to every solution throughout both procedures to prevent transcriptional changes that could impact downstream results. The remaining cells were washed several times in HBBS culture media before undergoing immunopanning. Cells were resuspended in 0.02% BSA in PBS and incubated at room temperature on a 10cm petri dish with anti-CD11b and secondary antibody already adhered to the dish (Thermo Scientific, 14-0112-82 and Jackson 112-005-167) for 10-15 minutes. After incubation the unadhered cells were washed away and remaining cells scraped off the plate directly into Trizol (Zymo Research R2050-1-200) and stored at -80 C until later use. RNA was extracted from isolated brain myeloid cells using the Qiagen RNeasy Kit (#74106) according to the manufacturer's guidelines. Bulk RNA-sequencing libraries were created for each sample using the Takara SMART-Seq mRNA HT LP kit (#634792) according to the manufacturer's protocol and recommendations.