Assessing the impact of Ccnc or Cdk8 loss +/- ATR inhibition on mRNA gene transcription using RNA-seq

We have found that loss of Ccnc or Cdk8 (members of the RNAPII mediator complex) provide ATRi resistance. As part of invesitgations into the mechanism of this suppression, we probed for specific changes in the transcriptome by performing RNA-seq in WT, Cdk8 KO and Ccnc KO mESCs following DMSO or ATRi (AZD6738) treatment. However, this did not identify any transcripts that were significantly differentially regulated following AZD6738 treatment in both Ccnc and Cdk8 KO, but not WT, cells. Similarly, pathway analyses of proteins whose transcripts were up- or down-regulated in a treatment-independent manner in both Cdk8 and Ccnc KO clones compared to WT mESCs did not provide a clear explanation why the Ccnc/Cdk8 KO cells are ATRi resistant. Two WT mESC biological replicates, three independent Ccnc KO mESC clones (D1, G2, H5) and three independent Cdk8 KO mESC clones (B9, H4, C8) were treated for 4h with DMSO or 900 nM AZD6738. The same WT samples were independently multiplexed with Ccnc KO samples, and again with Cdk8 KO samples, due to limitations on the number of samples that could be multiplexed, and to eliminate the impact of sequencing-run bias. The corresponding WT samples were used in downstream analysis.