GSM7750509: APOE4, Ctl_1; Mus musculus; RNA-Seq
Mice were aged to 9 months of age. Upon tissue harvest, mice were euthanized and perfused with 1U/ml heparin in PBS. Left hemisphere of the brains were dissected out and flash frozen on dry ice. Flash frozen brain tissue was homogenized in a Dounce homogenizer in Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.025% NP-40), and incubated on ice 15 minutes. The suspension was filtered through a 30 um filter to remove debris and pelleted at 500 x g 5 min at 4o C. Nuclei were washed and filtered twice with Nuclei Wash (1% BSA in PBS with 0.2 U/uL RNasin (Promega)). Nuclei pellets were resuspended in 500 uL Nuclei Wash and 900 uL 1.8 M Sucrose. This 1400 uL mixture was carefully layered on top of 500 uL 1.8 M sucrose and centrifuged at 13,000 x g 45 min 4oC to separate the nuclei from myelin debris. The nuclei pellet was resuspended in Nuclei wash at 1200 nuclei/uL and filtered through a 40 um FlowMi Cell Strainer. Isolated single nuclei were subjected to droplet-based 3' end massively parallel single-cell RNA sequencing using Chromium Single Cell 5' Reagent Kits as per manufacturer's instructions (10x Genomics) The generated scRNAseq libraries were sequenced using an Illumina sequencer.