Supplementary Figure 3: RNA-seq analysis of depletion of NCoR/SMRT co-repressors in OSKM reprogramming.

(a) Heatmap showing all genes from the RNA-seq analysis of MEFs transduced with OSKM and shRNAs against Luciferase, Ncor1,and Ncor2. Fold change is relative to MEFs. MEF and ESC data are taken from GSE29278 (same for panels c, d, f). (b) GO analysis showing the up-regulated genes from panel a on day 5 (left panel) and day 9 (right panel). (c) Heatmaps of the genes significantly differentially up-regulated or down-regulated in response to Ncor1/2 knockdown. Genes significantly up- or down-regulated in either Ncor1 or Ncor2 shRNA experiments were merged. (d) Boxplots of the genes in panel c and Fig. 1e, including MEF and ESC data. In general, Ncor1 and Ncor2 up-regulated genes on day 5 of OSKM reprogramming are specific to MEFs. The number of genes is indicated by ‘n’, the boxplots indicate the upper and lower quartiles, the central line is the median, the whiskers indicate 1.5*interquartile range and the circles are outliers. (e) Heatmap showing the fold-change of selected MET and epithelial-to-mesenchymal transition (EMT)-related genes from the RNA-seq analysis. (f) ‘Second wave’ genes, which are associated with the later stage of reprogramming, were mapped onto the reprogramming RNA-seq data. Gene expression was converted into a gene-wise Z-score, and P values were from a two-tailed Mann-Whitney-U test. The number of genes is indicated by ‘n’, the boxplots indicate the upper and lower quartiles, the central line is the median, the whiskers indicate 1.5*interquartile range and the circles are outliers. The 95% confidence intervals for day 5 data: shLuc (−0.44, −0.32), shNcor1 (−0.48, −0.35), and shNcor2 (−0.47, −0.36). And for day 9 data: shLuc (−0.16, −0.01), shNcor1 (0.058, 0.21), and shNcor2 (0.039, 0.18). (g, h) RT-qPCR for the indicated somatic genes on day 5 (panel g) and pluripotency genes on day 9 (panel h) in MEFs transduced with OSKM and shRNAs against Luciferase, Ncor1, Ncor2, or combined Ncor1 and Ncor2 in serum+Vc. Data are the mean ± s.e.m. (n=3 biological replicates for somatic genes and n=4 for pluripotency genes with 3 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test, * indicates P value <0.05. (i) RT-qPCR for the indicated pluripotency genes on day 13 of MEFs transduced with OSKM and shRNAs against Luciferase, Ncor1, Ncor2, and combined Ncor1 and Ncor2 in serum+Vc, measured relative to OSKM plus shLuc. MEFs and E14 ESCs are included for comparison. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test, * indicates P value <0.05.