GSM9396789: ctx-37-60-92; Mus musculus; RNA-Seq
Nuclei were isolated by pooling frozen brain tissue from samples of the same genotype. All procedures were performed on ice or at 4°C in an RNase-free environment. Briefly, frozen mouse brain cortex tissue was gently lysed in 3 mL of homogenization buffer (250 mM sucrose, 150 mM KCl, 30 mM MgCl₂, 60 mM Tris, 0.01% v/v Triton X-100, 0.001% v/v Digitonin, 0.01% v/v NP40, 1 mM DTT), supplemented with 0.2 U/mL RNase inhibitor (NEB, M0314) and a complete protease inhibitor cocktail (Roche, 11697498001). Lysis was performed using a Wheaton Dounce Tissue Grinder (30 strokes with pestle B). The lysate was filtered through a 40 µm cell strainer and centrifuged at 1,000 × g for 8 minutes to collect a nuclear pellet. To remove debris, the pellet was resuspended in 350 µL of homogenization buffer, mixed 1:1 with 50% iodixanol buffer (iodixanol 60% v/v in a buffer containing 250 mM sucrose, 150 mM KCl, 3 mM MgCl₂, 60 mM Tris), and layered over 600 µL of 29% iodixanol buffer (iodixanol 29% v/v in the same buffer). The sample was centrifuged at 13,500 × g for 20 minutes. The supernatant was discarded, and the nuclei were gently resuspended and washed in 1 mL of 1% BSA/PBS. Nuclear integrity and complete lysis were visually confirmed. Nuclei were manually counted and diluted to a concentration of 1,000 nuclei/µL in 1% BSA/PBS. Single-nucleus RNA-seq libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10X Genomics), following the manufacturer's instruction. Briefly, single nucleus gel-bead-in-emulsions (GEMs) were generated in Chromium Controller with NextGEM Chip G (10X Genomics). The first-strand cDNA was synthesized on-beads, followed by clean-up and amplification. The amplified cDNAs were examined in Bioanalyzer with High-Sensitivity DNA Chip (Agilent). Ten-ul of cDNAs were forwarded to the library preparation. The dual-indexed libraries were pooled and sequenced on the Illumina NovaSeq 6000 or NovaSeq X Plus sequencer (Illumina).