The published Methods state that the murine co-culture and TGFβ-iTreg conditions used activation medium containing 20 U/mL IL-2. However, the treatment metadata for the RNA-sequencing samples deposited under GSE272646 state that the organoid co-cultures received 20 U/mL IL-2, whereas the TGFβ-iTreg comparator received 300 U/mL. Could the authors clarify the actual IL-2 concentration used for each RNA-sequencing group and provide the contemporaneous sample and media-preparation records? If the concentrations differed, an IL-2-matched analysis or control appears necessary because IL-2 exposure could contribute to the PCA separation and differential-expression results attributed to organoid-derived factors.
The RNA-sequencing Methods also identify NM_016701.3 as the mm10 transcriptome reference, although this accession corresponds to the single mouse Nes transcript. Please provide the exact transcriptome build and annotation release used for alignment. Clarification would also be helpful for Figure 5, whose caption omits the statistical test and describes the expansion-index panel as a “survival curve,” and for the reproducibility of the 141-gene TCGA survival analysis.