Transcriptome-wide mapping of RNA:protein interactions of SRSF6 in HeLa cells grown under normoxic and hypoxic conditions by iCLIP

We used HeLa cells that express GFP-tagged SRSF6 at physiological levels and subjected them to 4 h or 24 h hypoxia (0.2% oxygen). Cells grown in normal conditions were used as control (Normoxia, 21% oxygen). We performed iCLIP using anti-GFP antibodies and compared the binding pattern of SRSF6 between normoxia, 4h hypoxia and 24h hypoxia. 3 biological replicates per condition were subjected to the iCLIP procedure with UV-crosslinking at 254 nm. For IPs either an anti-GFP antibody was used. No UV treatment served as negative control.

本研究使用在生理水平表达GFP标记SRSF6的HeLa细胞，将其分别置于氧浓度为0.2%的低氧环境中处理4小时或24小时；以正常培养条件（氧浓度21%，常氧（Normoxia））下生长的细胞作为对照组。随后采用抗GFP抗体开展iCLIP实验，对比常氧、4小时低氧及24小时低氧三种条件下SRSF6的RNA结合模式。每个实验条件均设置3次生物学重复，所有样本经254 nm紫外紫外交联后实施iCLIP流程；免疫沉淀（IP）实验仅使用抗GFP抗体，未进行紫外处理的样本作为阴性对照。