RIP sequencing in PANC-1 cells to map the YBX1-binding mRNA of PANC-1 cells treated with CAF-derived Evs

Perineural invasion (PNI) is a unique biological feature of pancreatic cancer and is a key cause of pancreatic cancer metastasis, recurrence and poor postoperative survival, but its mechanism is largely unclarified. Clinical sample analysis and endoscopic ultrasonographic elasticity scoring indicated that cancer-associated fibroblasts (CAFs) are closely related to the occurrence of PNI. Furthermore, CAF-derived extracellular vesicles played an extremely important role in PNI in a dorsal root ganglion (DRG) coculture model and sciatic nerve model. Next, we demonstrated that CAFs promoted PNI via extracellular vesicle transmission of PNI-associated transcript (PIAT). To explored the potential mRNA interacted with YBX1, we we performed YBX1 RNA immunoprecipitation–sequencing (RIP-seq). PANC-1 cells were pretreated with CAFs-derived Evs and collected for YBX1 RIP-seq. Briefly, target antibodies were immobilized on protein A/G magnetic beads for 30 min at room temperature. Then cell lysate was added into the mixture and incubated overnight at 4 °C. Finally, the precipitated RNAs were purified for deep sequencing.