GSM7651087: WT, hippocampus, 1y, replicate 2; Mus musculus; RNA-Seq
Mice were euthanized by carbon dioxide gas overdose, and the brains were rapidly dissected out. The hippocampus of 3-, 9-, and 12-month-old mice, the septum of 6-month-old mice, and the Hb of 3-, 9-, and 12-month-old were collected from 5xFAD mice and WT mice. The samples were cut into small pieces, placed in digestion media (Neurobasal media containing 1 mg/mL Collagenase type 1, 2, 4 (Worthington Biochemical Corp., United States) or papain (Worthington Biochemical Corp., United States), and 20 U/mL of DNaseⅠ (Sigma)), and incubated in a shaking incubator at 37 °C for 45 min. The digested tissues were mechanically dissociated and filtered through a 40 µm cell strainer. The single-cell suspension was centrifuged at 500 g for 15 min at 4°C and the resulting pellet was suspended in ice cold neurobasal media containing B27. This neurobasal media wash was repeated twice with two different spins at 100 g and 200 g centrifugation to remove dead cells and cellular debris. The single-cell suspension was finally suspended in an ice cold Hanks' balanced salt solution with 0.04 % bovine serum albumin (A3294, Sigma-Aldrich, United States) at approximately 2 ⅹ 105 cells/mL cell population. The single cells were profiled using inDropTM barcoded beads in a home-made microfluidic device connected to the Drop-seq apparatus in the Korea Brain Research Institute. Libraries from isolated single cells were generated according to the inDropTM Protocol with the following modifications (Zilionis et al., 2017). Amplified cDNAs were not fragmented, and cDNA cleanup and size selection were performed using SPRIselect beads (B23317, Beckman Coulter, United States). Amplified cDNAs and final libraries were assessed on an Agilent BioAnalyzer using the High Sensitivity DNA Kit (Agilent Technologies, United States). The libraries were pooled and sequenced on a NovaSeq 6000 system / a NextSeq500 system at two sequencer companies (Macrogen, Korea / LAS Science, Korea).