GSM5293725: MY1950; Mus musculus; OTHER
For DNA extraction from single dissected tumors to generate libraries for Tuba-seq, targeted sequencing of selected oncogenes. For bulk lung samples, following homogenization and overnight protease K digestion, genomicDNA was extracted from the lung lysates using standard phenol-chloroform and ethanolprecipitation methods. Q5 High-Fidelity 2x Master Mix (New England Biolabs, M0494X) was used to amplify the sgID-BC region from 50 ng of DNA from dissected tumors or 32 μg of bulk lung genomic DNA. The unique dual-indexed primers used were Forward: AATGATACGGCGACCACCGAGATCTACAC- 8 nucleotides for i5 index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-6 to 9 random nucleotides for increasing the diversity-GCGCACGTCTGCCGCGCTG and Reverse: CAAGCAGAAGACGGCATACGAGAT-6 nucleotides for i7 index-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-9 to 6 random nucleotides for increasing the diversity-CAGGTTCTTGCGAACCTCAT. The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter, A63881) using a double size selection protocol. The concentration and quality of the purified libraries were determined using the Agilent High Sensitivity DNA kit (Agilent Technologies, 5067-121791801811821831841851861871881891901911921931941951961974626) on the Agilent 2100 Bioanalyzer (Agilent Technologies, G2939BA). The libraries were pooled based on lung weights to ensure even reading depth, cleaned up again using AMPure XP beads, and sequenced (read length 2x150bp) on the Illumina HiSeq 2500 or NextSeq 500 platform (Admera Health Biopharma Services).