GSM8702683: APP/PS1, aCSF, replicate 1, snRNASeq; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane, and their brains were dissected. Hippocampi were dissected from brain, immediately frozen in liquid nitogen and stored at −80°C The frozen hippocampal tissues were homogenized with prechilled Dounce homogenizer with ice-cold homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH [pH 7.8], 1 mM dithiothreitol, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors, 5 μg/mL actinomycin, 0.32% Nonidet P-40, and 0.04% bovine serum albumin). After 25 strokes with a loose pestle, the homogenate was mixed 1:1 with OptiPrep, and the solution was centrifuged at 10,000 × g for 20 min at 4°C. The pellet of separated nuclei was washed to remove the OptiPrep and resuspended in DMEM/F12 supplemented with 10% fetal bovine serum. The nuclei were counted with a hemocytometer and then diluted to 400 nuclei per microliter. All buffers and gradient solutions for nuclear extraction contained 60 U/mL RNasin (Promega). the snRNA-seq libraries were generated using a Chromium Single Cell 3′ Library Kit v3 (10x Genomics, 1000078) according to the manufacturer's instructions. The concentrations of the final libraries were measured by Qubit (Thermo Fisher Scientific), and fragment lengths were determined using Fragment Analyzer (Advanced Analytical Technologies). The libraries were subjected to paired-end sequencing.