GSM6329930: APP_Microglia, IL33, 24hours; Mus musculus; RNA-Seq
In brief, we deeply anesthetized adult mice using isoflurane and then perfused them with ice-cold PBS. We isolated the forebrain, minced them into small pieces, then mechanically dissociated them with a Dounce homogenizer on ice. We used a Percoll gradient (30%; Sigma-Aldrich) to remove myelin. We blocked the resultant mononuclear cell suspensions with an FcR blocker for 10 min on ice, then incubated them with antibody in the dark for 30 min on ice. Next, we performed flow cytometry analysis and cell sorting with a BD Influx Cell Sorter. We used unstained controls to identify cell populations and visualized clear subpopulations of living microglia on scatter plots; the purity of microglial isolation was routinely >90% according to a reanalysis of the sorted cells. We generated scRNA-seq libraries using the Chromium Next GEM Single Cell 3′ Library Kit (v3.1 Chemistry; 10x Genomics) according to the manufacturer's instructions. In brief, we counted sorted living CD11b+ cells on a hemocytometer. We mixed single-cell suspensions (400 cells/µL) with reverse-transcription reagent mix and loaded them into the chip for single-cell encapsulation. We immediately incubated the encapsulated cells in a thermocycler for reverse transcription. We obtained barcoded cDNA and used it for library construction according to the manufacturer's instructions. We quantified the concentrations of the final libraries with Qubit (Thermo Fisher Scientific) and fragment lengths with Fragment Analyzer (Advanced Analytical). We performed paired-end sequencing of the libraries on a NovaSeq 6000 instrument (Novogene) according to the manufacturer's instructions.