Mutational Analysis of Organoid-Derived DNA

High molecular weight genomic DNA from the colonic, hepatic, and pulmonary organoids was extracted using RecoverEase DNA Isolation Kit (Agilent Technology, United States), according to the manufacturer’s instructions. Lambda EG10 phages were rescued using Transpack Packaging Extract (Stratagene, La Jolla, CA, United States). The gpt mutation assay was performed according to previously described methods (Fukai et al., 2018 (link)). Briefly, E. coli YG6020 was infected with the phage and spread on M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG) and incubated for 72 h at 37°C to select colonies with a plasmid carrying the gene encoding chloramphenicol acetyltransferase, as well as mutated gpt. The 6-TG–resistant isolates were cultured overnight at 37°C in LB broth containing 25 mg/ml of Cm, harvested by centrifugation (7,000 rpm, 10 min), and stored at −80°C. Mutational spectra of 6-TG coding sequences were determined using PCR and direct sequencing, and a 739-bp DNA fragment containing gpt was amplified by PCR as described previously (Fukai et al., 2018 (link)). Sequence analysis was performed using Eurofins Genomics software (Tokyo, Japan).