Quantifying DNA Lesion Repair in CRC

The TLS assay was previous described [12 (link)]. Briefly, 200 ng of competitor gapped plasmid and the 50 ng of lesion-containing plasmid were transfected into HT29 and SW480 cells using Lipofectamine2000 (ThermoFisher). Cells were incubated at 37 °C for 4 h and harvested. Subsequently, DNA was extracted using DNA isolation kit (QIAGEN) and transformed into the recA- E. coli to propagate closed plasmids for 16 h. Plasmid DNA was extracted from E. coli culture and lesion region was amplified by PCR (forward primer: 5′ TTGTACTGAGAGTGCACCATGCCCGT-3′, reverse primer: 5′-GAGTCAGTGAGCGAGGAAGCGTGCTG-3′). Restriction enzymes XhoI and SphI (NEB) were used to digest the PCR products, which were next subjected to TSQ Altis™ Triple Quadrupole Mass Spectrometer (ThermoFisher) to determine the nucleotides originally repaired in CRC cells.