NextSeq 500 sequencing; Gene expression profiling of TROP2+ cells from human CRC (colorectal carcinoma) specimens
Gene expression profiling of TROP2+ cells from human CRC (colorectal carcinoma) specimens
Freshly resected human colon tumor specimens were digested using Dispase to obtain single cell suspension. Cells were stained with following antibodies: CD45-PacificBlue, CD31-PacificBlue, EpCAM-Phycoerythrin, and TROP2-Allophycocyanin; shortly before sorting, Hoechst33358 was added to the cells. Two populations were sorted from each tumor using fluorescence activated cell sorting (FACS): Hoechst-/CD31-/CD45-/EpCAM+/TROP2+ and Hoechst-/CD31-/CD45-/EpCAM+/TROP2- cells. Total RNA was isolated using RNeasy Micro Kit (Qiagen) as described in the manufacturerâ€™s protocol. Sequencing libraries were prepared using Takara Smarter Stranded Total RNA-seq Kit v2 Pico Input Mammalian (PN:634413) according to the manufacturer's protocol.