RNA-seq of single cholangiocyte-derived organoids reveals high organoid-to-organoid variability

Organoids have been established from the majority of tissue resident stem cells. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such a heterogeneity has not been performed before. Here, we used RNA-seq of individual organoids to address this question. Importantly, we find that batch-to-batch variation is very low, even when prepared by different researchers. On the other hand, there is organoid-to-organoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Overall design: Methods: Picking single organoids followed by bulk-RNA-seq with UMI libraries to assess heterogeneity. The impact of microenvironment (culturing method) on gene expression of organoid cultures by RNA-seq.The impact of passaging in gene expression. Dataset 1: RNA-seq data of 35 cholangiocyte-derived organoids from liver tissue of four 3-months-old mice, only one liver for each set, Set 1: 8, Set 2: 10, Set 3: 5 , Set 4: 12 Dataset 2: RNA-seq data of three 3-months-old cholangiocyte-derived organoid from liver tissue cultures, generated by pooling digested material from different animals each (Pool A = 2 mice; Pool B = 4 mice; Pool C = 3 mice), cultured in either matrigel domes or shaking suspension (in total 6 samples). Dataset 3: RNAseq of 3 organoid sets (1, 3 and 4) in passage 4 and the same organoid sets in passage 11 (in total 6 samples- each sample consists of 2 technical replicates).