NextSeq 500 sequencing; Gene expression profiling of Trop2+ cells in mouse Apc-deficient small intestinal early adenomas
Gene expression profiling of Trop2+ cells in mouse Apc-deficient small intestinal early adenomas
Adult Rosa26-tdTomato Apc-flox/flox Lgr5-CreERT2 mice were intraorally administered with 1 mg tamoxifen (in EtOH/mineral oil) and sacrificed by cervical dislocation after 6 weeks. The small intestinal epithelium contained tumors with endogenous expression of the red fluorescent protein tdTomato. A single cell suspension was prepared from whole ileum using dispase digestion. Cells were stained with the following antibodies: Hoechst33358, anti-CD45-PacificBlue, anti-EpCAM-FITC, anti-Trop2-APC. Using fluorescent activated cell sorting (FACS), we isolated live epithelial cells (Hoechst/CD45-/EpCAM+) and classified them into three groups: tdTomato-negative cells (mostly differentiated cells), Trop2+ tdTomato+ cells and Trop2- tdTomato+ cells. Total RNA was isolated using RNeasy Micro Kit (Qiagen) as described in the manufacturer's protocol. The sequencing libraries were prepared using SMARTer Stranded Total RNA-seq Kit v2 Pico Input Mammalian following the manufacturer's instructions.