GSM9400254: WT+antiPD1_2; Mus musculus; RNA-Seq
Fresh tumor tissue is minced and enzymatically dissociated (e.g., Collagenase IV/DNase I, 37°C, 15–45 min), followed by mechanical disruption and filtration (70 µm → 40 µm). After RBC lysis (if needed), cells are washed, debris/dead cells are removed (e.g., FACS or density gradient), and viability (>85%) is confirmed. Cells are resuspended in scRNA-seq buffer (700–1,200 cells/µL) and immediately processed for library prep (e.g., 10x Chromium) or stabilized in RNA preservation buffer. Maintain cold conditions and minimize processing time to preserve RNA integrity. Isolated tumor cells (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.