seCLIP-Seq (single-end enhanced crosslinking and immunoprecipitation) of STAT3, OCT4, and SRSF1 in TE03 (I3) human embryonic stem cells (hESCs). seCLIP-Seq (single-end enhanced crosslinking and immunoprecipitation) of STAT3, OCT4, and SRSF1 in 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".

本研究采用单端增强交联免疫沉淀测序（seCLIP-Seq，single-end enhanced crosslinking and immunoprecipitation），对TE03人胚胎干细胞（human embryonic stem cells, hESCs）中STAT3与OCT4转录因子的体内RNA结合位点进行定位分析。作为对照参照，我们还针对经典RNA结合蛋白SRSF1在该细胞中开展了seCLIP实验。STAT3-RNA复合物的免疫沉淀实验设置了2次独立生物学重复，同时包含1份尺寸匹配输入（size-matched input, SMInput）样本与1份非交联对照样本。OCT4与SRSF1的seCLIP实验均设置2次生物学重复，并以2份尺寸匹配输入文库作为对照。文库在Illumina HiSeq 2500测序平台上完成测序。本数据集配套论文题为《解析人胚胎干细胞多能性网络中的RNA结合蛋白图谱》（Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells）。论文摘要：胚胎干细胞（embryonic stem cell, ESC）的自我更新与细胞命运决定由大量分子信号共同驱动。尽管人类胚胎干细胞（human embryonic stem cells, hESCs）中的转录调控因子已被广泛研究，但RNA结合蛋白（RNA-binding protein, RBP）对人类多能性的贡献程度仍不明确。本研究通过蛋白质组范围的筛选，在hESCs中鉴定出810种RNA结合蛋白。研究发现，大量RBPs在hESCs中优先表达，并在干细胞早期分化过程中呈现动态调控模式。值得注意的是，近200种RBPs的表达受多能性核心调控因子OCT4的敲低影响，其中数十种可被该因子直接结合。通过交联免疫沉淀测序（CLIP-Seq，cross-linking and immunoprecipitation），本研究证实多能性相关的STAT3与OCT4转录因子可在hESCs中与RNA结合，并验证了STAT3与保守长链非编码RNA NORAD的结合。综上，本研究结果表明，RBPs在人类多能性中的作用比此前认知的更为广泛。