GSM8969450: Liver, AAV8-GFP, western diet; Mus musculus; RNA-Seq
To isolate nuclei, frozen livers from 3 mice randomly selected in each group were diced in NP40 lysis buffer (IGEPAL® I3021, Sigma-Aldrich, USA), dounce homogenized, and incubated on ice for 5 minutes. The homogenate was filtered using a 70-µm cell strainer and then a 40-µm cell strainer, and centrifuged for 5 minutes at 500 x g at 4°C. The supernatant was removed and the nuclei pellets were resuspended in wash buffer (1% bovine serum albumin, 0.2U/µL RNAse inhibitor) and marked with Nuclear Pore Complex HTOs (Biolegend) to identify individual mice from each pool prior to FACS-purification using the dye SYTOX™ AADvanced™ (ThermoFischer, USA) to remove debris. Following collection, nuclei integrity was visually verified with Trypan blue staining and nuclei were counted. Nuclei from individual mice were pooled at equal ratio prior to encapsulation. Encapsulation was performed using the 10X Chromium Controller using 3' GEMs kits v3.1 with groups run in separate lanes. Libraries were sequenced targeting ~25k reads/nuclei.