title: GSM6329927: APP_Microglia, IL33, 24hours, rep3 [BulkRNA_IL33_24h_3]; Mus musculus; RNA-Seq
library_construction_protocol: 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 sorted approximately 10,000 living microglia directly into RA1 Lysis Buffer and stored them at −80 °C until RNA extraction. We then extracted RNA from the microglia using a NucleoSpin RNA XS Kit (Macherey-Nagel) according to the manufacturer's instructions. We immediately converted the extracted RNA into full-length complementary DNA (cDNA) using a SMART-Seq v4 kit (Takara Bio). Next, we quantified the concentrations of the cDNA libraries with Qubit (Thermo Fisher Scientific) and fragment lengths with Fragment Analyzer (Advanced Analytical). For library construction, we tagmented 1 ng cDNA by incubation with Tn5 transposase (Illumina) for 30 min at 55 °C and then amplified the tagmented cDNA by PCR for 12 cycles. We size-selected the libraries (100–600 bp) with AMPure XP beads (Beckman Coulter). 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 using a NextSeq 500 or NovaSeq 6000 instrument (Novogene) according to the manufacturer's instructions.