Fig. 6: RNA-seq reveals transcriptional changes in PRDM14-deficient hESCs and hPGCLCs.

a Volcano plot showing down- (log2FC < (−0.5), padj < 0.05) and upregulated (log2FC > 0.5, padj < 0.05) genes in hPGC-competent hESCs upon PRDM14 loss. PRDM14-AID hESCs were treated with IAA for one passage (3 days) and sorted as AP⁺ cells for RNA-seq along with “no IAA” control. b Volcano plot showing down- (log2FC < (−0.5), padj < 0.05) and upregulated (log2FC > 0.5, padj < 0.05) genes in hPGCLCs upon PRDM14 loss. PRDM14-AID hPGCLCs were specified with or without IAA for 4 days and sorted as NANOS3-tdTomato⁺AP⁺ cells for RNA-seq along with “no IAA” control. c Boxplot overlaying down- (log2FC < (−0.5), padj < 0.05) and upregulated (log2FC > 0.5, padj < 0.05) genes upon PRDM14 knockdown in conventional hESCs (microarray data from¹³) and RNA-seq analysis in PRDM14-AID-depleted PGC-competent (4i) hESCs from this study. The distance from the bottom to the upper line of the box spans the interquartile range (IQR), the horizontal line in the box indicates the median of the dataset, while the whiskers show ±1.5× IQR. Note that the majority of differentially expressed genes show consistent changes. d Principal component analysis (PCA) of hPGCLC RNA-seq samples. PRDM14-depleted hPGCLCs (cl11 and cl21; highlighted in red) separate from the other samples along principal component 1 (PC1). e qPCR validation of selected DEGs from RNA-seq. hPGCLCs from cl11 and cl21 were sorted as NANOS3-tdTomato⁺AP⁺ cells, while soma denotes the double-negative population from the same experiments. Strongly upregulated genes are plotted separately. Data show gene expression relative to no IAA hPGCLCs and normalised to GAPDH, mean ± SEM of n = 5 for SOX17 and VENTX, mean ± SEM of n = 3 independent experiments for NANOS3, BLIMP1, TFAP2C, KLF4 or n = 2 for other genes. Statistical significance of hPGCLC no IAA vs. +IAA was assessed by multiple unpaired t-tests yielding two-tailed P values: KLF4 **(P = 0.0054), NANOG **(P = 0.0053), NANOS3 *(P = 0.0452), VENTX ****(P < 0.0001). The parental (“no TIR1”) control is shown in Supplementary Fig. 6D. f Gene ontology (GO) analysis on down- and upregulated genes in PRDM14-deficient hESCs. Top non-redundant GO terms are shown as −log10(p value). g GO analysis on down- and upregulated genes in PRDM14-deficient hPGCLCs. Top non-redundant GO terms are shown as −log10(p value). For complete list of GO terms see Supplementary Data 6.