Kras-ex3op Amplicon; Tumor 88; 129S Kras-(ex3op/ex3op) urethane-induced Mus musculus Lung Tumor
Dissected tumors were homogenized and RNA isolated using RNABee (Fisher). Phase-lock heavy gel (Fisher) maximized recovery. RNA was co-precipitated with Pellet Paint (Novagen) using isopropanol. Maximal RNA volumes were used for reverse transcription (50% of total reaction volume) with M-MuLV reverse transcriptase (NEB) according to manufacturer instructions with addition of oligodT primers and RNAseOUT (Invitrogen) to generate cDNA.  Primers with Ion Torrent adapter sequences and bar codes corresponding to tumor sample and allele amplified (native or ex3op, see Supplementary Table 7 of manuscript) were used for PCR amplification. PCR reactions utilized Platinum Taq (Invitrogen), the supplied buffer (final concentration 0.7x), 1 mM dNTPs, 1 mM MgCl2, 0.4 µM primers, and 5% of total reaction volume contributed by cDNA. Reactions were run with 40 cycles as follows: denaturation at 94ºC for 30 seconds, step-down annealing at 65-50ºC for 45 seconds, and elongation at 72ºC for 2-minutes. 450 bp products were gel purified and pooled for sequencing.