title: GSM9131565: STING_Control_2; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions were filtered through 40 μm cell strainers and resuspended into PBS containing 0.05% BSA following the 10X Genomics protocol. Isolated tumor single cell suspension (700–1,200 cells/µL in PBS + 0.04% BSA) are loaded onto a microfluidic platform (e.g., 10x Genomics Chromium) for single-cell encapsulation and barcoding. Reverse transcription is performed using template-switching oligos (TSO) to generate cDNA, followed by amplification and fragmentation. Indexed libraries are prepared via end-repair, A-tailing, and adapter ligation, then pooled and quantified (e.g., Bioanalyzer/Qubit). Sequencing is performed on an Illumina platform (e.g., NovaSeq) with recommended read lengths (e.g., 28 bp Read 1, 91 bp Read 2, 8 bp index). Quality control (QC) includes assessing cDNA yield, library size distribution, and doublet rates. Store libraries at –80°C until sequencing. Raw sequencing reads (FASTQ files) are processed using Cell Ranger (10x Genomics) or similar tools (e.g., STARsolo, Alevin) for alignment (to GRCh38/GRCm38), barcode/UMI correction, and gene counting. Low-quality cells are filtered (mitochondrial reads <20%, gene counts 200–6,000/cell, UMI >500). Data normalization (e.g., SCTransform, LogNormalize) and integration (Harmony, Seurat) are applied to correct batch effects. Dimensionality reduction (PCA, UMAP) and clustering (Louvain, Leiden) identify cell populations, followed by differential gene expression analysis (Wilcoxon, MAST). Cell types are annotated using reference databases (SingleR, Azimuth with manual correction). Results are visualized and exported for downstream analysis.