Purpose: Organoids are gaining more traction as physiologically relevant models for host physiology, but their transcriptional stability has not been identified over long term culture. Comparing this to its host-derived tissue and a transformed cell line of this intestinal location could provide insight into the stability and maintenance of cellular programs in vitro. Methods: Jejunum organoids were derived from 2 5-week old piglets, and grown separately in triplicate for 3 and 12 weeks. Tissue homogenate, organoids from both time points (divided in triplicate at start of experiment to monitor culture-dependent variability) and three IPEC-J2 passage rates were isolated for RNA using an RNEasy mini kit following manufacturers protocols. Results: RNA was enriched for PolyA and sequenced on an illumina sequencer. Reads were checked for quality, trimmed, and mapped following the tuxedo suite pipeline (modified to fit our parameters) and CLC Genomics Workbench to identify unknown gene locations. Conclusions: Organoids cultured for 12 weeks showed strong inter- and intra variability, congruence between independent cultures, and strong epithelial resemblance to host tissues. It further exemplified a more extensive transcriptome than IPEC-J2, showing more physiologically relevant genes
