Sanger sequencing for mutation screening

RNA was purified from cell pellets using the RNeasy® Mini Kit (QIAGEN, 74104) according the manufacturer’s protocol. To eliminate possible genomic DNA contaminations the RNA samples were treated with DNA-free^TM DNA Removal Kit (Ambion®, Life Technology, AM1906). Complementary DNA (cDNA) was synthesized from RNA templates using the RETROscript® Kit (Ambion®, Life technology, AM1710). To amplify Tp53 and Kras sequences form cDNA and genomic DNA for sequencing as templates gene specific primers were designed (see Supplementary Table 6) and purchased from Eurofins, Martinsried, Germany. PCRs were carried out using Platinum Taq High Fidelity DNA polymerase (Invitrogen, 11304–029) and 10 mM dNTP Mix (PCR Grade, Invitrogen, Life Technologies, 18427–088). Trp53 and Kras PCR products were sent for Sanger sequencing to external companies (GATC Biotech, Constance, Germany and LGC Genomics, Berlin, Germany). Received sequences were screened for mutations by alignment with corresponding wild-type sequences (Serial Cloner, SerialBasics and BLAST, NCBI) and analysis of sequencing histograms employing Chromas Lite software (Technelysium).