PolyA RNA-seq from HCT116 (WT and CDK8as/as) cells in normoxia or hypoxia and treated with DMSO or 3MB-PP1

To determine the impact of CDK8 kinase activity on steady-state mRNA levels, we performed RNA-seq analysis of colorectal carcinoma cell line HCT116 (CDK8wt/wt and CDK8as/as) in the presence of absence of 3MB-PP1 to specifically inhibit analog senstitive (as) CDK8. PolyA RNA for two independent biological replicates was purified from HCT116 cells (CDK8wt/wt and CDK8as/as) incubated in normoxic (ambient oxygen) or hypoxic (1% oxygen) conditions for 24 hrs and treated with DMSO or the ATP-analog 3MB-PP1, and was sequenced on the Ion Torrent Proton platform. Reads were aligned (GSNAP) to the human genome (hg19) and gene-level counts (HTseq-count) were used for differential expression analysis (DESeq2).

为探究CDK8激酶活性对稳态mRNA水平的影响，我们针对结直肠癌细胞系HCT116（CDK8野生型纯合[CDK8wt/wt]与类似物敏感型纯合[CDK8as/as]）开展RNA测序（RNA-seq）分析，设置了3MB-PP1存在与不存在的两组实验，以特异性抑制类似物敏感型（as）CDK8。我们将两份独立生物学重复的HCT116细胞（CDK8wt/wt与CDK8as/as）分别置于常氧（常压氧气）或低氧（1%氧气）条件下孵育24小时，随后用二甲基亚砜（DMSO）或ATP类似物3MB-PP1进行处理，之后纯化其聚腺苷酸RNA（polyA RNA），并在Ion Torrent Proton测序平台上完成测序。测序读段通过GSNAP比对至人类参考基因组hg19，利用HTseq-count获取基因水平计数数据，并通过DESeq2开展差异表达分析。