title: GSM6586242: Isolated single nuclei from hippocampus of 9-month-old P301S-APOEKO mouse treated with antibiotic cocktail; Mus musculus; RNA-Seq
library_construction_protocol: Mice were randomly assigned to antibiotic cocktail (4 mg/mL kanamycin, 0.35 mg/mL gentamicin, 8,500 U/mL colistin, 2.15 mg/mL metronidazole, 0.45 mg/mL vancomycin) or water group. Antibiotic cocktail (100 uL) or water were administered daily for one week at 2 weeks of age via oral gavage. Mice were then aged to 40 weeks. 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.