Searching for the fusion transcripts created during proteotoxic stress

To verify the hypothesis that proteotoxic stress induces the formation of fusion transcripts in non-cancerous and cancerous cells derived from breast epithelium, we exposed the cells to heat shock (HS, 1h at 43 degrees). We selected MCF10A non-tumorigenic breast epithelial cells (isolated from the mammary gland with fibrocystic disease) and MCF7 breast adenocarcinoma cells (estrogen receptor-positive, ER+). Global transcriptome analysis was performed in untreated cells and at 2h and 4h after HS. The TimeLapse-Seq method was used, which allows for obtaining information about newly produced RNA in relation to total RNA in one sequencing reaction [1]. To metabolically label the newly produced RNA, 4-thiouridine (4sU, a thymidine analog) was added to the medium for the duration of the experiment. To convert 4sU to cytidine analogs (providing U-to-C mutations that enable tagging of new transcripts when sequenced), samples were treated with 2,2,2-trifluoroethylamine (TFEA) and sodium periodate (NaIO4) as described in [1]. All libraries were sequenced on the Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 200M reads/sample. Obtained data were subjected to the standard RNA-seq data analysis procedure, including the additional steps required for TimeLapse-Seq, described in [1]. Identification of gene fusions was performed using the Arriba tool [2]. [1] Schofield JA. et al. Nat Methods. 2018, 15:221-225. [2] Haas BJ. et al. Genome Biol. 2019, 20:213.