GSM9578787: Mouse glioma sample, DMSO treatment, replicate 1; Mus musculus; RNA-Seq
Nuclei were isolated from fresh-frozen mouse brain tissues using Dounce homogenization (D8938, Sigma-Aldrich) and the Nuclei EZ Prep Kit (Nuc101-1KT, Sigma-Aldrich) according to published protocols. Nuclei were centrifuged at 300 g for 10 minutes at 4°C and resuspended in FACS buffer (1% BSA in PBS) containing Recombinant RNase Inhibitor (2 U/mL; Takara) and anti-CD16/CD32 Fc block (BD Biosciences). After incubation for 5 minutes, nuclei were stained with NeuN antibody (ab190565, Abcam) and incubated on ice for 30 minutes with gentle shaking. Nuclei were centrifuged again at 300 g for 10 minutes at 4°C and resuspended in FACS buffer containing RNase inhibitor. Hoechst 33342 dye was added prior to sorting. NeuN-positive and NeuN-negative nuclei were sorted separately using fluorescence-activated cell sorting (FACS) into DNA LoBind tubes containing final buffer (200 µL UltraPure BSA, Thermo Fisher; 800 µL PBS; and 5 µL Protector RNase Inhibitor). Approximately 50,000 nuclei were collected per sample and adjusted to a concentration of ~1 × 10^6 nuclei/mL. Single-nucleus RNA-seq libraries were generated using the Chromium Single Cell 3′ Library & Gel Bead Kit v3.1 (10x Genomics) following the manufacturer's protocol. Nuclei suspensions were adjusted to target approximately 10,000 nuclei per sample and loaded onto the Chromium Controller (10x Genomics) to generate Gel Bead-in-Emulsions (GEMs). Reverse transcription, cDNA amplification, and library construction were performed according to the Chromium Single Cell 3′ Library Construction protocol v3.1. Library quality was assessed using an Agilent Bioanalyzer at the Stanford Protein and Nucleic Acid Facility. Multiplexed libraries were sequenced by Novogene (Sacramento, USA) on an Illumina NovaSeq S4 platform using paired-end 150 bp sequencing, targeting approximately 100 million reads per library.