GSM9651908: brain, in vivo, methanol-fixed; Mus musculus; RNA-Seq
Option 1, for unfixed-control samples: Nuclei were extracted using the Boyou Nuclear Isolation Kit (BoHao Biotechnology, Cat. 52201-10). ≤50 mg of tissue was homogenized in 600 μL lysis buffer, incubated on ice for 4 min, filtered, and centrifuged at 500 ×g for 5 min at 4°C. Nuclei were resuspended in 50 μL washing buffer, assessed for integrity using trypan blue and AO/PI staining, and filtered through a 40 μm cell strainer. Samples were kept on ice throughout the process. Option 2, for methanol-fixed samples: Nuclei were extracted using the Boyou Nuclear Isolation Kit (BoHao Biotechnology). Following extraction, pre-chilled 100% methanol was added dropwise to the nuclei suspension to a final concentration of 80%, and samples were incubated at -20°C for 30 minutes. Before library construction, nuclei were processed using the SeekMate Cell/Nucleus Fixation and Preservation Kit (SeekGene, Cat. K02301-0803). The suspension was centrifuged at 1000×g for 5 min at 4°C, resuspended in 400 μL Buffer Q, mixed with 800 μL WB3 buffer, and assessed for integrity. Single-nucleus suspensions were processed using the SeekOne DD Single-Cell 3' Transcriptome Kit (SeekGene, K00202-0202) according to the manufacturer's instructions. The resulting cDNA was PCR-amplified, and libraries were generated using the SeekOne DD Single Cell RNA Library Prep Kit (SeekGene, K00202-0204).