RNA-Seq to validate the effect of substrate and format as drivers of transcriptional variance in human intestinal organoids

An initial comparison of RNA-sequencing datasets from 251 experiments on 35 human intestinal organoid lines revealed that differences in transcriptional signatures could be influenced by variations in the format (3D, monolayers on transwells, and monolayers on 96-well plates) as well as substrate (collagen and Matrigel) in which the organoid lines are cultured. While the organoid lines were a common to these datasets, there were differences in experimental approaches in different laboratories. To rule out potential confounding variables, we performed an RNA-Seq experiment using 5 jejunal organoid lines cultured at the same time in different formats and substrates to evaluate their roles in baseline transcriptional signatures and determine if key findings from the initial analysis could be validated. Overall design: 25 samples were analyzed (no technical replicates). The cell lines used were J2,J3, J4, J6, J11. Each of these cell lines were plated as follows: 3D on Matrigel; Transwells on Matrigel; Transwells on collagen; Monolayers on Matrigel; Monolayers on collagen