For sc-RNA-seq, tissues were minced and dissociated using 0.1% Type I Collagenase (Sigma, C0130) and 10 μg/mL DNase I (Sigma, 11284932001) in PBS with 5% FBS at 37°C with shaking (50 r.p.m.) for 40 min, with intermediate collection every 20 min. Cell suspensions were filtered (40 μm) and treated with Red Blood Cell Lysis Solution (Thermo Fisher, 00-4333-57). For single nucleus sequencing, the nuclear fraction was isolated from frozen tissue specimens using the Shbio Nuclei Isolation Kit (SHBIO, #52009-10, China) following standardized procedures. Tissue samples were rapidly homogenized in chilled lysis buffer using a mechanical tissue disruptor until complete liquefaction was achieved. The resulting homogenate was filtered through a 40 μm nylon mesh to remove cellular debris and particulate matter, followed by centrifugation at 500 × g for 5 minutes at 4°C to pellet nuclei. Subsequent density gradient separation was performed by sequential addition of PB1, PB2, and PB3 solutions (as defined by the Shbio Nuclei Isolation Kit protocol), with intact nuclei accumulating at the PB2-PB3 interface. Isolated nuclei were gently resuspended in 50 μL of NB buffer using pipette mixing and quantified using an automated cell counting system (Thermo Fisher Scientific, USA). For sc-RNA-seq, cDNA synthesis, barcoding, and library preparation were performed using the Chromium Next GEM Single Cell 3' Kit according to manufacturer's protocol (CG000731). Libraries were quantified using High Sensitivity DNA Chip (Agilent) and Qubit High Sensitivity DNA Assay (Thermo Fisher), then sequenced on NovaSeq 6000 (Illumina) with 2×150 bp chemistry.  For single-nucleus RNA sequencing, purified nuclei were processed immediately using the Chromium Single Cell 3' Library and Gel Bead Kit v3 (10X Genomics, USA) on a Chromium Controller instrument (10X Genomics, USA) for single-nucleus barcoding and cDNA synthesis. Library preparation was conducted according to the manufacturer's standardized protocol, with final sequencing performed on an Illumina NovaSeq 6000 platform (Illumina, USA) to generate high-throughput sequencing data.