GSM8794721: 5xFAD_vehicle_1mo_rep1; 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 magnetic activated cell sorting according to the manufacturers (Miltenyi Biotec) guidelines. Samples were resuspended in PB buffer (0.5% BSA in PBS pH 7.2) with CD11b MicroBeads (Miltenyi Biotec # 130-093-634) and incubated for 15 minutes at 4° C protected from light. Cells were then washed and resuspended in PB buffer before being placed on an LS column (Miltenyi Biotec #130-042-401). Magnetic separation was repeated on a second LS column to maximize purity. After separation, cells were resuspended in Trizol and stored at -80° C for 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.