Next Generation Sequencing  of circRNAs in bladder cancer derived ITGA6+EVs
EVs served as crucial mediators preferably achieve their biological effect in recipient cells to create a supportive pre-metastatic niche via delivering distinct biomolecules, among which circular RNAs (circRNAs) with high stability and abundance in tumor derived EVs were under extensive exploration. Moreover, it is well-established that circRNAs play a significant role in BCa lymphatic metastasis. To explore the crucial circRNAs in BCa derived ITGA6+EVs, the ITGA6+EVs from total UM-UC-3-EVs were isolated by affinity capture using anti-ITGA6 magnetic beads and the circRNA expression profile was investigated using next-generation sequencing (NGS). Overall design: We isolated ITGA6+EVs from total UM-UC-3-EVs by affinity capture using anti-ITGA6 magnetic beads and degrades the abundant linear RNAs with RNase R. Then, each sample was amplified and transcribed into fluorescent cRNA utilizing a random priming method. Double-stranded cDNA fragments are subjected to end-repair and A-tailing. After amplification by PCR, the reaction system and program for circularization are configured and set up. The concentration and specific activity of circRNAs were measured by DNBSEQ-G400.