GSM8972101: 5xFAD Control; Mus musculus; RNA-Seq
Briefly, frozen cortex tissue was homogenized in 0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8. The buffers were supplemented with 10 μl/ml RNasin, Superasin, and EDTA-free protease inhibitor cocktail. 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, and 10% donkey serum. 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 Library prepared according to manufacurer's instructions (single cell 3' GEX, 10x Genomics). 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) analysis and then sequenced in paired-end mode on a HiSeq 2500 instrument (Illumina, San Diego, CA) targeting a depth of 50,000–100,000 reads per nucleus/cell.
GEO
GSM8972101_r1