title: GSM8716844: WT mice and TAM treatment (CfD-TAM); Mus musculus; RNA-Seq
library_construction_protocol: Nuclei isolation and sorting of microglia via fluorescence-activated nuclei sorting (FANS) were performed as previously described. Hippocampi from three individual mice per experimental group were pooled for nuclei isolation. Dissected hippocampi were homogenized in nuclei isolation buffer (0.5% Triton X-100, 0.1M Sucrose, 5mM MgCl2, 1mM EDTA, 10mM Tris-HCl) supplemented with 40U/μl RNasin using a Dounce homogenizer. Nuclei were pelleted (700 g for 5 minutes at 4C), resuspended in 2 mL of nuclei isolation buffer with RNasin, and incubated for 10 minutes on ice. Following incubation, nuclei were pelleted (700 g for 5 minutes at 4C), washed once with sort buffer (1X HBSS, 10% bovine serum albumin, 1mM EDTA, and 40U/μl RNasin) and then resuspended in 260 μL of sort buffer with RNAsin. Nuclei were stained for 30 minutes on ice with the following antibodies: anti-PU.1-PE (1:100, BioLegend, 681308) and anti-Olig2-647 (1:2500, Abcam, ab225100) for microglia and oligodendrocytes, respectively. Nuclei were labeled with DAPI (1:1000, BioLegend, 422801). Following staining, nuclei were pelleted (700 g for 5 minutes at 4C), resuspended in 500 μL of sort buffer with RNAsin, and strained through a 70 μm mesh strainer. PU.1+ singlets were sorted on a Beckman Coulter Mo-Flo Astrios EQ into sort buffer with RNasin. After sorting, BSA was added to a final concentration of 1%. Nuclei were pelleted at 800 g for 10 minutes at 4C and then resuspended in PBS with 1% BSA. Individual nuclei were then coupled to beads using the 10X Genomics Chromium controller. Single nuclei sequencing libraries were prepared following the 10X Genomics Chromium GEM-X Single Cell 3' V4 kit protocol. Libraries were paired end sequenced on an Illumina NovaSeq X Plus platform (28X10X10X90X) to a minimum depth of 100,000 reads per nuclei.