Transcriptomic profiling of genetically engineered mouse tumors using SOCRATES
Transcriptomic profiling of genetically engineered mouse tumors using SOCRATES
Tumor tissues were collected from genetically engineered mouse models (LCas, VAKP, VAKPS) following colonoscopy-guided submucosal injection of lentivirus encoding SOCRATES constructs. Tumors were dissected at defined timepoints, processed into fragments, and enzymatically dissociated using the Miltenyi Tumor Dissociation Kit (#130-095-929) on the gentleMACS Octo Dissociator. Single-cell suspensions were filtered through 100 μm cell strainers and processed for downstream single-cell RNA sequencing. Total RNA was captured directly during single-cell encapsulation using the 10x Genomics Chromium Single Cell 3' v3.1 platform. No separate nucleic acid extraction was performed. Cell hashing antibodies (TotalSeq-B, BioLegend) were used for sample multiplexing and demultiplexing. Single-cell libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) according to the manufacturer's instructions. This protocol generated one gene expression (GEX) library and one hashtag oligo (HTO) library per reaction. Cells were stained and sorted for viability prior to encapsulation.