2.4. Validation of detected transcript fusions with Sanger sequencing

Validation of novel transcript fusions was performed with the use of Sanger's direct sequencing method on the 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA) with ABI PRISM 1.1 BigDye Terminator Cycle Sequencing Ready Reaction Kit (Life Technologies). Before sequencing, RNA (200 ng) was converted to cDNA with the Omniscript RT Kit (Qiagen GmbH). The reaction was carried out for 1 hour at 37°C in a volume of 20 μL using a mixture of 1× concentrated buffer (2 μL; Omniscript RT Kit), 5× concentrated dNTPs (2 μL; Omniscript RT Kit), 4 U/μl RT‐O polymerase (1 μL; Omniscript RT Kit), 50 μM random nonamers (1.6 μL), and 1× concentrated RiboLock RNAse inhibitor (1 μL; Fermentas Thermo Fisher Scientific, Waltham, MA). Obtained cDNA was used as a template in PCR reactions, in which each amplicon was amplified using specific primers designed with the Primer3 Input software available on the website http://frodo.wi.mit.edu/ (for primer sequences and annealing temperatures used in PCR reactions, see Supporting Information Table S2). Each amplicon contained sequences of both rearranged genes. The PCR products were visualized by electrophoresis in a 2% agarose gel in the presence of bromide ethidium (0,3 μL/mL), cleaned with Exo I (volume 0.6 μL; concentration 10 U/μl; Life Technologies) and Sap (volume 0.6 μL; concentration 2 U/μl; Boehring Manheim GmbH, Germany; Life Technologies) enzymes mixture according to manufacturer's recommendations and then sequenced as described earlier.