title: GSM8196538: 5xFADEif2ATRAP_snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Microglial nuclei were isolated based on the eGFP-L10a fluorescence of newly formed ribosomes in the microglia nucleoli as described in Ayata et al. Briefly, one male and one female mouse for each sample were sacrificed by cervical dislocation. Brain regions were quickly dissected and homogenized in 0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8 with a glass Dounce homogenizer (1984-10002, Kimble Chase, Vineland, NJ). The buffers were supplemented with 10 μl/ml RNasin, Superasin, and EDTA-free protease inhibitor cocktail (11836170001, Roche). The homogenate was then spun through a 29% iodixanol cushion. The resulting nuclear pellet was resuspended in 0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, supplemented with 10 μM DyeCycle Ruby (V10304, Invitrogen), and 10% donkey serum (017-000-121, Jackson Immunoresearch, West Grove, PA). Microglial nuclei were sorted in a BD FACS Aria cell sorter by gating for the lowest DyeCycle Ruby, which indicates nuclei singlets and a high GFP signal. For single nuclei RNA sequencing, isolated nuclei were used immediately for 10X sequencing Libraries were constructed from isolated microglial nuclei (at least 5,000 nuclei) using the Chromium platform (10x Genomics, Pleasanton, CA) with the 3′ gene expression (3′ GEX) V2 kit, using a targeted input of ~8,000 nuclei per sample. In brief, gel-bead in emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs using Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly-A-tailing, adaptor ligation, and 10X-specific sample indexing following the manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (Thermofisher)