title: GSM8950670: VHL treated APPPS1-21 cortex 4, snRNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: Single nuclei suspensions were generated from the cortices of vehicle and antibiotic treated APPPS1-21 male mice using the 10x Genomics Chromium Nuclei Isolation Kit (#PN-1000494). Briefly, frozen brain cortex samples (approximately 40 mg per sample) were transferred to pre-chilled sample dissociation tubes and 500 uL of lysis buffer was used to dissociate tissue for 10 minutes. Dissociated tissue was pipetted onto nuclei isolation columns and centrifuged for 16,000 x g for 20 seconds at 4°C. Flowthrough from the columns were vortexed for 10 seconds to resuspend nuclei and centrifuged at 500 x g for 3 minutes at 4°C. Supernatant was removed and pellets were resuspended with 500 uL of debris removal buffer and centrifuged at 700 x g for 10 minutes at 4°C. The top myelin layer was taken off using a transfer pipette. The rest of the supernatant was then removed, and pellets were resuspended in 1 mL of wash and resuspension buffer and centrifuged at 500 x g for 5 minutes at 4°C. This process was then repeated once. Then samples were resuspended in wash and resuspension buffer and centrifuged at 300 x g for 8 minutes at 4°C Lastly, supernatant was removed, and nuclei pellets were resuspended in 150 uL of wash and resuspension buffer and vortexed for 3 seconds. Final nuclei concentration was determined using the Invitrogen Countess 3 automated cell counter. Further processing of nuclei was completed using 10X Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 chemistry workflow The single nucleus suspension was created using the 10x Genomics Chromium Nuclei Isolation Kit (#PN-1000494). After isolation, nuclei were loaded into the 10x Genomics Chromium Controller with a target of 10,000 nuclei per sample. Gel bead-in emulsions (GEM) generation and library preparation were performed according to the 10X Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 chemistry workflow. Libraries were pooled and sequenced on an Illumina Novaseq 6000.