Cell lines were harvested, centrifuged and stored at -80°C. Cells were grown in RPMI1640 medium supplemented with 10% tetra-cycline free foetal bovine serum, 2 mM L-Glutamine and 100 IU/ml penicillin/streptavidin at 37°C in a 5% CO2 humid atmosphere Cells were treated with JQ1, THZ1, the combination or left untreated and 10h later, cells were harvested. Total RNA was extracted using miRNeasy kit (Qiagen) according to manufacturer's instructions, including on-column DNase treatment, and concentration was determined with the Nanodrop (Thermo Scientific). 100 ng of RNA from cells was used to perform an Illumina sequencing library preparation using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to manufacturer's protocol. During library preparation 15 PCR cycles were used. Size distribution and quality was evaluated with a high sensitivity DNA ChIP on the bio-analyzer (Agilent) and qPCR quantification of the libraries using the Illumina Kapa Library quantification kit (Lightcycler 480 qPCR mix Kapa).
Experimental Factor: compound
THZ1 (35 nanomolar)