Transcriptional readthrough and splicing upon chemotherapy treatment [long-read, nascent RNA]

The Neugebauer lab has previously shown that splicing occurs co-transcriptionally and is coupled to 3'end cleavage. It is known that stress, such as hyperosmotic shock, affects RNA processing. The cellular stress response is an important component of cancer cell response to chemotherapy that can determine cell survival. Herein, we study how the coupling of the RNA processes in leukemia cells is affected in response to stress and therapeutic agents. We used short- and long-read sequencing to follow RNA processing upon treatment. Overall design: To determine changes in RNA processing steps such as transcriptional readthrough and co-transcriptinal splicing we used long-read sequencing. We analyzed nascent RNA isolated from chromatin fraction of cells, that was additionally depleted of polyA-RNA and rRNA. Before isolation of nascent RNA the cells were treated with: - 1uM Imatinib (IM) for 1h and 18h - 5uM JTE-607 (JTE) for 1h - 110mM KCl (KCL) for 45min. All time points ended at the same time and thus the cells were collected in paralell with the untreated control cells (CTRL and CTRLim). Each experiment was repeated three times (1, 2, 3)