title: GSM9012232: MC38_Vector; Mus musculus; RNA-Seq
library_construction_protocol: For single-cell RNA sequencing (scRNA-seq) of subcutaneous tumors, a stable MC38 cell pool expressing SPP1 or a control vector was established via lentiviral transduction. Western blotting was performed to confirm successful expression of SPP1. These cells were then subcutaneously injected into C57BL/6 mice for tumor formation. Seven days later, mice in the OE-SPP1 and OE-vector groups were intraperitoneally injected with anti-PD1 (10 mg/kg, twice a week). After two weeks of treatment, the mice were euthanized, and tumor tissues were carefully excised. The tumor tissues were dissociated into single-cell suspensions using enzymatic digestion (e.g., collagenase and DNase), followed by mechanical dissociation. The resulting cell suspension was filtered through a 70 µm cell strainer to remove large debris and dead cells. Single-cell libraries were prepared using nova X plus, which allows for high-throughput single-cell capture and barcoding. The captured cells were encapsulated into droplets, where reverse transcription and cDNA amplification occurred. After library construction, sequencing was performed on a high-throughput platform (nova X plus), with a minimum depth of 20,000 reads per cell.