ena-STUDY-TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY-04-04-2021-14:17:29:076-628
seCLIP-Seq (single-end enhanced crosslinking and immunoprecipitation) of STAT3, OCT4, and SRSF1 in TE03 (I3) human embryonic stem cells (hESCs)
Here, we employed seCLIP-Seq (single-end enhanced crosslinking and immunoprecipitation) to map the in vivo RNA-binding sites of the STAT3 and OCT4 transcription factors in TE03 human embryonic stem cells (hESCs). As a reference for comparison, we also performed seCLIP on these cells for the classical RNA-binding protein SRSF1. Immunoprecipitation of STAT3-RNA complexes was carried out in two independent biological replicates, along with one size-matched input (SMInput) sample and a non-cross-linked control. OCT4 and SRSF1 seCLIP experiments were performed in two biological replicates and two size-matched input libraries were used as a control. Libraries were sequenced on the Illumina HiSeq 2500 platform. This data accompanies the manuscript: "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".
Here, we employed seCLIP-Seq (single-end enhanced crosslinking and immunoprecipitation) to map the in vivo RNA-binding sites of the STAT3 and OCT4 transcription factors in TE03 human embryonic stem cells (hESCs). As a reference for comparison, we also performed seCLIP on these cells for the classical RNA-binding protein SRSF1. Immunoprecipitation of STAT3-RNA complexes was carried out in two independent biological replicates, along with one size-matched input (SMInput) sample and a non-cross-linked control. OCT4 and SRSF1 seCLIP experiments were performed in two biological replicates and two size-matched input libraries were used as a control. Libraries were sequenced on the Illumina HiSeq 2500 platform. This data accompanies the manuscript: "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".