title: GSM8657356: cortical brain tissue, 192_GD, snRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Fresh cortical tissue were dissected from left hemisphere of 12 month old WT, Gba, SNCA tg and Gba-SNCA mice after euthanasia. Single nuclei were isolated as previously described with modifications5. All procedures were carried out on ice or at 4oC. Briefly, fresh cortical tissue was homogenized in 8.4 ml of ice-cold nuclei homogenization buffer [2 M sucrose, 10 mM Hepes (pH 7.5), 25 mM KCl, 10% glycerol, 1 mM EDTA (pH 8.0), and ribonuclease (RNase) inhibitors freshly added (40U/ml)] using a Wheaton Dounce tissue grinder (10 strokes with the loose pestle and 10 strokes with the tight pestle). The homogenate was carefully transferred into a 15 ml ultracentrifuge tube on top of 5.6 ml of fresh nuclei homogenization buffer cushion and centrifuged at 25,000 rpm for 60 min at 4°C in an ultracentrifuge. The supernatant was removed, and the pellet was resuspended in 1 ml of nuclei resuspension buffer [15 mM Hepes (pH 7.5), 15 mM NaCl, 60 mM KCl, 2 mM MgCl2, 3 mM CaCl2, and RNase inhibitors freshly added (40U/ml)] and counted on a hemocytometer with Trypan Blue staining. The nuclei were centrifuged at 500g for 10 min at 4°C with a swing bucket adaptor. They were subsequently resuspended at a concentration of 700 to 1200 nuclei/μl in the nuclei resuspension buffer for the next step of 10x Genomics Chromium loading and sequencing Droplet-based single nucleus RNA sequencing and data alignment: The snRNA-seq libraries were prepared by the Chromium Single Cell 3′ Reagent Kit v3.1 chemistry according to the manufacturer's instructions (10x Genomics). The generated snRNA-seq libraries were sequenced using Illumina NovaSeq6000 S4 at a sequencing depth of 300 million reads per sample. For snRNA-seq of brain tissues, a custom pre-mRNA genome reference was generated with mouse genome reference (available from 10x Genomics) that included pre-mRNA sequences, and snRNA-seq data were aligned to this pre-mRNA reference to map both unspliced pre-mRNA and mature mRNA using CellRanger version 3.1.0.