Searching for the fusion transcripts created during proteotoxic stress
Searching for the fusion transcripts created during proteotoxic stress
Non-tumorigenic human breast epithelial MCF10A cells (authenticated in 2016) were cultured in DMEM/F12 medium supplemented with 5% horse serum (BioWest, Nuaillé, France), 5 µg/mL insulin (Sigma-Aldrich, Saint Louis, MO, USA), 0.5 µg/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (Sigma-Aldrich). Breast cancer MCF7 cells (authenticated in 2016) were cultured in DMEM/F12 medium supplemented with 10% Fetal Bovine Serum (EURx, Gdansk, Poland). Cells were seeded on culture plates the day before the start with HS stimulation. Control and HS-treated cells were continuously exposed to 4sU, with the concentration depending on the analysis time: 200nM for samples analyzed 2h after HS and 100nM for samples analyzed 4h after HS (3h and 5h of exposure, respectively). Total RNA isolation was performed at appropriate time points. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). The isolated RNA from each sample was chemically treated using 2,2,2-trifluoroethylamine (TFEA) and sodium periodate (NaIO4) to  convert incorporated 4sU to the C analog. The experiment was performed in four biological replicates, and RNA from individual replicates was pooled before sequencing. The ribosomal RNA (rRNA) is depleted for target transcript enrichment. The fragmented RNA undergoes reverse transcription into cDNA. Strand-specific libraries were prepared and sequenced.