GSM8338535: Mr BMT-treated CSF1RWT/I792T, replicate1, scRNA-seq; Mus musculus; RNA-Seq
The cell suspension was primarily obtained according to our previous protocol29. In brief, the mice were perfused with 0.9% saline and an inhibitor cocktail solution (actinomycin D 5 μg/mL, triptolide 10 μM, anisomycin 27.1 μg/mL), after which the brains of the mice were quickly sliced into 200 μm sections. Next, the sections were incubated in choline chloride solution supplemented with 10 μM NBQX (Tocris, Cat#: 0373), 50 μM APV (Tocris, Cat#: 3693), an inhibitor cocktail, and an influx of oxygen (95% O2 and CO2) for 30 min at 37 °C. Afterward, the sections were digested in 20 U/mL papain (Worthington, LK003178) and 100 U/mL DNase I (Sangon, B100649-0040) for 20 min at 37 °C, and then the sections were incubated in 1 mg/mL protease (Sigma–Aldrich, P5147) and 1 mg/mL dispase (Worthington, LS02106) for 20 min at 25 °C. Next, the sections were gently and repeatedly blown through a pipette into single-cell suspensions and then filtered through 40 μm cell strainers. The cell debris was removed by 30% Percoll (Millipore, P1644) in DPBS supplemented with 0.5% BSA. Finally, the cells were resuspended in DPBS supplemented with 0.5% BSA. Approximately 24,000 cells for each group (N = 3 mice) were loaded for 10x Genomics library preparation. A cocktail of transcriptional and translational inhibitors was included in the cell preparation to prevent ex vivo microglial reactivation28,29. Single-cell libraries were constructed using a Chromium controller and Chromium single-cell 3 reagent version 2 kit (10x Genomics). Then, the libraries were subjected to a 150 bp paired-end sequencing by HiSeq X Ten (Illumina).