Fig. 3

Elevated activity of miR-182 promotes SI lineage formation in part through post-transcriptionally suppressingSOX2. (A) Nascent transcriptional (ChRO-seq) and steady-state expressional (RNA-seq) changes of transcription factors critical for SI lineage specification (transition from DE to Duo). # defined as unstable genes in Fig. 2D. $ defined as stringent unstable genes in Fig. 2G. (B) miRNAs that are enriched during SI lineage specification (adjusted P < 0.05 and absolute log2 foldchange > 0.5 and by DESeq2 Wald test, RPMMM > 1000 at least in one stage of a two-group comparison) and have conserved target sites on EOMES, LHX1 or SOX2 (TargetScan). (C) smRNA-seq showing a significant upregulation of miR-182 during SI lineage specification. ** Adjusted P < 0.01 by DESeq2 Wald test in smRNA-seq. DE, n = 3; Duo, n = 6. (D) qPCR showing a significant upregulation of miR-182 during SI lineage specification in both hESC and iPSC cell line. ** P < 0.01 by two-tailed t-test. N = 2–3 samples per cell line per stage. (E) qPCR showing a significant downregulation of SOX2 during SI lineage specification in both hESC and iPSC cell line. *** P < 0.001 by two-tailed t-test. N = 2–3 samples per cell line per stage. (F) Reverse Spearman correlation between miR-182 and SOX2 during 5-day window of DE cells receiving SI induction cocktail. N = 2–3 samples per day. (G) qPCR of miR-182 levels in iPSC directed differentiated cells receiving treatment of mock, scrambled control or miR-182 inhibitors. **** P < 0.0001 by two-tailed t-test. (H) qPCR of SOX2 in iPSC directed differentiated cells receiving treatment of mock, scrambled control or miR-182 inhibitors. ** P < 0.01 by two-tailed t-test. N = 3–6 per condition across 2 independent experiments. DE, definitive endoderm. Duo, duodenal spheroid. RPMMM, reads per million mapped to miRNAs. RQV, relative quantitative value