GSM8901925: adjacent normal cells, patient_07; Homo sapiens; RNA-Seq
All sample collection procedures complied with the regular routine in clinical practice. Tumor tissues and matched normal mucosa (≥5 cm from tumor margins) were collected from 18 patients (36 total samples). Fresh surgical specimens (≥100 mg/sample) were processed as follows: (1) Immediate transfer to pre-chilled RNase-free PBS (Ca²⁺/Mg²⁺-free) collection tubes maintained at 4°C; (2) Removal of blood clots and adipose tissue under sterile, ice-cold conditions; (3) Minced into 0.5 mm³ fragments using micro-scissors; (3) Triple-washed with pre-cooled PBS to eliminate debris. Fragments were enzymatically digested in dissociation buffer (0.35% collagenase IV, 2 mg/ml papain, 120 U/ml DNase I) at 37°C with 100 rpm agitation for 20 minutes. The reaction was quenched using PBS supplemented with 10% FBS. Cell suspensions were: (1) Mechanically dissociated via 10× pipetting with a 1 ml tip; (2) Sequentially filtered through 70 μm and 30 μm strainers; (3) Centrifuged at 300×g (4°C, 5 min); (4) Treated with 1× RBC Lysis Buffer (MACS 130-094-183) for 2–10 min at RT; (5) Labeled with Dead Cell Removal MicroBeads (MACS 130-090-101) for 15 min at RT; (6) Purified using MS Columns (130-042-201) with two additional centrifugation steps. Final viable cells were resuspended in 100 μl PBS containing 0.04% BSA. Cell viability (>85%) was verified by trypan blue exclusion. Concentrations (700–1,200 cells/μl) were quantified using a Countess II Automated Cell Counter (Thermo Fisher), meeting single-cell sequencing library preparation standards. Single-cell suspensions were processed using the Chromium Single-Cell 3' Kit (v3, 10×Genomics) on a Chromium microfluidic chip, targeting capture of 8,000 individual cells. Following manufacturer protocols, captured cells underwent cDNA amplification and library preparation. Single-Cell Transcriptomic Analysis: (1) Preprocessing with Seurat (v4.1.0): Initial filtering: Retained cells with >500 detected genes and mitochondrial gene content ≤25%; data normalization: LogNormalize method applied; dimensionality reduction: PCA performed using top 2,000 variable genes; top 20 principal components retained; batch correction: Harmony algorithm implemented for dataset integration; clustering & visualization: Cell clusters identified and projected in 2D space using UMAP. (2) Differential Expression Analysis: Marker genes identified via FindAllMarkers function with thresholds:Expressed in ≥10% of cluster cells; Log₂ fold-change ≥0.26; Benjamini-Hochberg adjusted p-value ≤0.01.