gDNA was isolated from 1 × 106 cecum cells with the Qiagen Genomic-tip 20/G Kit using the Qiagen Genomic DNA buffer set applying the protocol for tissues. TE buffer (20×, 200 mM Tris⋅HCl, 20 mM EDTA, pH 8.0) was used in all steps. gDNA was quantified via Pico Green standard curve (Quant-iT PicoGreen dsDNA reagent; Invitrogen) (56 (link)) using Lambda (λ)/HindIII DNA (Thermo Fisher Scientific). Fluorescence was measured in a plate reader (20-s shaking, 485-nm excitation, 520-nm emission), and DNA quality was assessed with electrophoresis.
In total, 3.5 ng of gDNA was used for Long Amplicon PCR (56 (link), 57 (link)). PCR was performed in 25-µL reactions containing 1× LongAmp Hot Start Taq2× Master Mix (New England Biolabs), 0.1 µM primer, and nuclease-free water. Temperature profile was used as follows: 94 °C, 2 min; 94 °C, 15 s; x °C (individual), 12 min; 72 °C, 10 min; 4 °C, forever. Annealing temperatures and cycle numbers were adjusted for 8.7-kb β-globin fragment (accession number X14061) to 64 °C and 25 cycles; for 6.6-kb DNA-polymerase β (accession number AA79582) to 65 °C and 24 cycles; for 10-kb long mitochondrial fragment to 64 °C and 17 cycles; for 117-bp short mitochondrial fragment to 60 °C and 18 cycles. Ten microliters of PCR product were quantified in duplicate using Pico Green and lesion burden was calculated according to Furda et al. (56 (link)).
In total, 3.5 ng of gDNA was used for Long Amplicon PCR (56 (link), 57 (link)). PCR was performed in 25-µL reactions containing 1× LongAmp Hot Start Taq2× Master Mix (New England Biolabs), 0.1 µM primer, and nuclease-free water. Temperature profile was used as follows: 94 °C, 2 min; 94 °C, 15 s; x °C (individual), 12 min; 72 °C, 10 min; 4 °C, forever. Annealing temperatures and cycle numbers were adjusted for 8.7-kb β-globin fragment (accession number X14061) to 64 °C and 25 cycles; for 6.6-kb DNA-polymerase β (accession number AA79582) to 65 °C and 24 cycles; for 10-kb long mitochondrial fragment to 64 °C and 17 cycles; for 117-bp short mitochondrial fragment to 60 °C and 18 cycles. Ten microliters of PCR product were quantified in duplicate using Pico Green and lesion burden was calculated according to Furda et al. (56 (link)).
