RNA-Seq after knockdown of the core pluripotency transcription factors OCT4, SOX2, and NANOG in TE03 (I3) human embryonic stem cells (hESCs)

The OCT4, NANOG and SOX2 transcription factors (TFs) are at the core of the pluripotency program, activating the expression of pluripotency genes and repressing lineage-specific genes, many of which are themselves TFs. However, the extent to which genes encoding RNA-binding proteins (RBPs) are also affected by the core transcriptional machinery is unknown. To address this, we employed siRNA to individually knockdown (KD) OCT4, SOX2, and NANOG in TE03 (I3) human embryonic stem cells (hESCs) and performed RNA-Sequencing of cells harvested at day six post-transfection. Cells treated with a non-targeting negative control siRNA were used as control. This experiment accompanies our manuscript titled: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that many RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell differentiation. Notably, nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Using cross-linking and immunoprecipitation (CLIP-Seq), we discover that the pluripotency-associated STAT3 and OCT4 transcription factors interact with RNA in hESCs and confirm the binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated".