title: GSM7791292: Scrambled control, snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Mouse tumors were macro-dissected from normal appearing brain and flash-frozen in liquid nitrogen. Tumor samples were removed from liquid nitrogen storage and nuclei were isolated from the frozen tissues using an adaptation of a previously established protocol. Briefly, each sample (two mice per condition were pooled for each sample) was placed on a clean petri dish and washed with 800 mL of Detergent-Lysis Buffer (0.1% Triton-X). A number 22 blade was used to dissociate tissue, which was then transferred into a 7 mL Dounce Homogenizer. Tissue was first homogenized with eight strokes of the loose Pestle, and then 12 strokes of the tight Pestle. The crude nuclei homogenate was then passed through a 40-micrometer strainer and spun down at 3,200 𝗑 g for 5 minutes at 4°C in a swing-bucket centrifuge. The pellet was resuspended in 1 mL of low-sucrose buffer and then gently layered above 4 mL of high sucrose buffer without disrupting the density gradient. The gradient was then spun down at 3,200 𝗑 g for 20 minutes at 4°C, and the ensuing pellet was resuspended in 25–150 mL of 0.04% BSA and 1 U/µL Sigma Protector RNase Inhibitor (3335399001) in 1X PBS. An aliquot was taken to be stained with Acridine Orange dye (1:10 dilution) and counted/imaged using a Luna-FL Dual-Fluorescence Cell Counter (Logos Biosystems). Following manufacturer's recommendations, the nuclei suspensions were next processed via the Single Cell Gene Expression Assay (10X Genomics). Nuclei were loaded onto a Next GEM Chip G (10X Genomics) targeting a yield of 5,000 nuclei. For all single-nuclei experiments, the Single Cell Gene Expression Kit (10X Genomics) was used according to the manufacturer's protocol recommendations. Library preparation was performed according to the manufacturer's instructions. Libraries were pooled and sequenced on the NextSeq 500 System (Illumina) using High-Output Reagent Kit v2.5 (150 cycles; Illumina). single nucleus RNA-sequencing