Time-lapse SLAM-seq for nuclear RNA

Xist RNA, which directs the process of X chromosome inactivation in mammals, localizes in cis over the chromosome from which it is transcribed, recruiting chromatin modifiers to silence underlying genes. To analyze cis-limited Xist RNA localization we have developed time-resolved super-resolution imaging of individual Xist RNA molecules in single cells. Using this approach, we quantify fundamental parameters underpinning Xist RNA dynamics, demonstrating a feedback mechanism linking Xist RNA synthesis and degradation, and an unusual coupling behavior that physically links temporally separated Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a separable function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique and unusual behavior of Xist RNA. SLAM-seq was used to assay Xist RNA's half-life. Initially, the cells expressing Xist were exposed to 4sU-containing medium for 4 hours, followed by 4sU washout and change back to normal medium. After 0min, 30min, 60min, 90min,120min, and 150min, harvest the cells, and extracts the nuclei becuase Xist RNAs were located in nuclei, to enrich the Xist signal. T2C signature were counted for each treated sample, and accordingly calculated the half-life for different RNA.