GSM8517178: NRAS/AKT Tumor, Isotype, scRNA-seq; Mus musculus; RNA-Seq
2g of tumor tissue were extracted from cancerous livers, cut into small pieces, and dissociated into cell suspensions using gentleMACS Dissociator (Miltenyi Biotec) in dissociation buffer containing 0.5mg/ml of Collagenase IV (Gibco) and 100μg/ml of DNase I (Goldbio) at 37℃. Single cell suspensions were collected after filtering through a 70μm cell strainer, and stained with fluorescent conjugated antibodies specific to CD45 (clone 30-F11, Biolegend) and TER-119 (Biolegend) for 20 min at 4°C. Live, single, tumour (CD45-TER119-) cells were then sort-purified with BD Influx into FBS-containing tubes, and were kept on ice until the cells were further processed for scRNAseq. Single cell encapsulation and cDNA libraries were prepared with Chromium Next GEM Single Cell 5' Reagent Kit v2 (Dual Index) and Chromium Next GEM Chip K Single Cell Kit, following manufacturer's instructions