GSM5769880
Brains were homogenized with Dounce homogenizer and microglia were enriched with Percoll solution (30%/70%). CD11bHigh/CD45Low) were collected using FACSDiva v8.0.1 (BD Biosciences) Single-cell RNA sequencing was performed on these samples using the Chromium platform (10x Genomics, Pleasanton, CA) with the 3' gene expression (3' GEX) V3 kit, using an input of ~10,000 cells. In short, Gel-Bead in Emulsions (GEMs) were generated on the sample chip in the Chromium controller, then barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter 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 NovaSeq instrument (Illumina, San Diego, CA) targeting a depth of 50,000-100,000 reads per cell. single cell RNA seq / Chromium Single Cell 3' Reagent Kits