NextSeq 500 sequencing; Gene expression profiling of Trop2+ cells in mouse Apc-deficient small intestinal hyperplastic epithelium
Gene expression profiling of Trop2+ cells in mouse Apc-deficient small intestinal hyperplastic epithelium
Adult Ki67-RFP Apc-flox/flox Villin1-CreERT2 mice were intraorally administered with 1 mg tamoxifen (in EtOH/mineral oil) and sacrificed by cervical dislocation after 7 days. In these mice, proliferating cells are marked by endogenous expression of the red fluorescent protein RFP. The hyperproliferating small intestinal epithelium was isolated from the proximal part of jejunum and the single cell suspension was prepared 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: RFP-negative cells (mostly differentiated cells), Trop2+ RFP+ cells and Trop2- RFP+ cells. Total RNA was isolated using RNeasy Micro Kit (Qiagen) as described in the manufacturer's protocol. The sequencing libraries were prepared using Stranded Total RNA-seq Kit v2 Pico Input Mammalian following the manufacturer's instructions .