GSM6788501: PT-6W; Homo sapiens; RNA-Seq
Tumor tissues or lymph nodes from mice were collected and minced in sterile PBS solution. Subsequently, each sample was digested with 0.1 mg/ml Liberase and 0.1 mg/mL DNAse I in PBS for 5-30 min at 37 °C with manual vortexing. The dissociation was terminated with DMEM containing 10% fetal bovine serum, and the samples were filtered successively through a 70-μm and 40-μm cell strainer to collect a suspension of single cells. The filtrate was centrifuged at 300×g for 5 min at 4 °C and washed twice with medium. Cells were stained for viability using Trypan Blue. In some experiments, primary cells were then plated in tissue culture flasks in medium containing puromycin to select tumor cells derived from primary tumor tissues and metastatic lymph nodes. The selected tumor cells were expanded ex vivo into sublines of PTs (derived from the primary tumors) or LNMs (derived from LNs with micrometastases) and then subjected to molecular and functional analysis. The single tumor cells were rinsed with DPBS on ice before loading into the Gel Bead & Multiplex Kit and Chip Kit (10× Genomics, Pleasanton, CA, USA) for the construction of a chromium single-cell 3′-library according to the manufacturer's instructions. Libraries were sequenced on an HiSeq X Ten system and mapped to the human reference genome (build GRCh38) using CellRanger-4.0.0 (10× Genomics).