Measurement of RNA kinetics in response to chronic hypoxia using 4sU labeling

The purpose of this project is to simultaneously measure RNA kinatics (RNA synthesis and degradation rates) on HCT116 cells calculated in chronic hypoxia condition. First, HeLa cells are cultured under normoxia or hypoxia condition during 36 hours, and subsequently the medium is replaced with which including 4-thiouridine (4sU). Total RNA is obtained from the labeled cells in time series. The total RNA are then alkylated by IAA causing T-to-C conversions to identify newly synthesized RNA. The sequence libraries of the IAA-treated samples were prepared with TruSeq stranded mRNA Sample Prep Kit (Illumine), and sequenced on the Nova-seq 6000 platform in 150-bp paired end. The RNA synthesis and degradation rates of individual genes are calcurated from the time series for the sequencing data of the 4sU-labeld RNAs.

本项目旨在在慢性缺氧（chronic hypoxia）条件下的HCT116细胞中，同步测定RNA动力学（RNA合成与降解速率）。首先将HeLa细胞置于常氧（normoxia）或缺氧（hypoxia）条件下培养36小时，随后将培养基更换为添加有4-硫尿苷（4-thiouridine，4sU）的培养液。通过时序采样获取标记细胞内的总RNA，再利用碘乙酰胺（IAA）对总RNA进行烷基化处理，诱导T-to-C碱基转换以区分新合成的RNA。采用TruSeq链特异性mRNA样本制备试剂盒（Illumina）构建经IAA处理样本的测序文库，并在Nova-seq 6000平台上开展150 bp双端测序。基于4sU标记RNA的测序数据时序信息，计算得到单个基因的RNA合成与降解速率。