title: Inducible deletion of DGAT1 and 2 from microglia exacerbates neurodegeneration and endolysosomal lipid accumulation in male PS19 mice
design: Inducible deletion of DGAT1 and 2 from microglia exacerbates neurodegeneration and endolysosomal lipid accumulation in male PS19 mice
library_construction_protocol: Frozen cortex from 3 mice of the same genotype were pooled as a single sample. Samples were selected based on those being closest to the mean values of hippocampal volumes. Tissue was homogenized using a Dounce homogenizer in 1 mL of lysis buffer (10 mM Tris-HCl, pH = 7.4; 10 mM NaCl, 3 mM MgCl2; 0.005% NP40; and 0.2 U/μl RNase Inhibitor in nuclease-free water at 4°C) and incubated on ice for 15 min. A 30 μm MACS SmartStrainer was used to remove cell debris and large clumps, followed by centrifugation at 500 x g for 5 min at 4°C. After carefully removing the supernatant, the nuclei pellet was resuspended with 5 mL Nuclei wash and resuspension buffer (1% BSA and 0.2 U/μl RNase Inhibitor in 1 X PBS). Then cell debris removal step, centrifugation, and resuspension were repeated 2 times. Only 500 μl of Nuclei wash and resuspension buffer was added into the last resuspension step. This solution was mixed with 900 μl of Sucrose Cushion Buffer I (2.7 mL Nuclei Pure 2M Sucrose Cushion Solution with 300 μl Nuclei Pure Sucrose Cushion Solution and then carefully layered to the top of 500 μl Sucrose Cushion Buffer I in a 2 mL Eppendorf tube. This sucrose gradient was centrifuged at 13,000 x g for 45 min at 4°C. After centrifugation, the nuclear pellet was resuspended by 500 μl Nuclei Wash and Resuspension Buffer. The nuclei concentration was determined using a Countess with pre-DAPI stain. Finally, nuclei concentration was adjusted to ∼1200 nuclei/μl using Nuclei Wash and Resuspension Buffer followed by proceeding to the 10 x Genomics protocol.  Tamoxifen (200 mg/kg) or corn oil were administered at 3 months of age via oral gavage once per day for 5 consecutive days. Isolated nulcei were subjected to droplet-based 3′ end massively parallel single-cell RNA sequencing using Chromium Single Cell 3′ Reagent Kits (10x Genomics) following the manufacturer's instructions. Isolated nulcei were subjected to droplet-based 3′ end massively parallel single-cell RNA sequencing using Chromium Single Cell 3′ Reagent Kits (10x Genomics) following the manufacturer's instructions.