Genome-Wide Adductomics Analysis Reveals Heterogeneity in the Induction and Loss of Cyclobutane Thymine Dimers across Both the Nuclear and Mitochondrial Genomes
Abstract
:1. Introduction
2. Results
2.1. The Effect of SSR Irradiation on HaCaT Cell Viability
2.2. Optimisation of DNA:Anti-T<>T MAb Ratio
2.3. DDIP-qPCR Quantification of the Induction and Repair of CPDs Induced by UVR
2.4. Nuclear and Mitochondrial Genome-Wide Mapping of T<>T Induction and Repair
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Cell Culture
5.2. Cell Preparation and Treatment
5.3. Cell Viability
5.4. DNA Extraction and Preparation
5.5. DNA Shearing
5.6. DNA Immunoprecipitation (DDIP) Assay
5.7. Removal of CPD Adducts Prior to PCR and Next-Generation Sequencing
5.8. Quantitative PCR Analysis
5.9. MicroPlex Library Preparation™ Kit for Next-Generation Sequencing
5.10. Bioinformatic Analysis of NGS Data
5.11. Quantification of mtDNA Damage
5.12. Quantification of mtDNA Content
5.13. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Availability of Materials and Data
References
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GAPDH Primer Sequence | |
Forward | 5′-GCCCCCGGTTTCTATAAATTG-3′ |
Reverse | 5′-GTCGAACAGGAGGAGCAGAGA-3′ |
Myoglobin exon 2 Primer Sequence | |
Forward | 5′-AAGTTTGACAAGTTCAAGCACCTG-3′ |
Reverse | 5′-TGGCACCATGCTTCTTTAAGTC-3′ |
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Alhegaili, A.S.; Ji, Y.; Sylvius, N.; Blades, M.J.; Karbaschi, M.; Tempest, H.G.; Jones, G.D.D.; Cooke, M.S. Genome-Wide Adductomics Analysis Reveals Heterogeneity in the Induction and Loss of Cyclobutane Thymine Dimers across Both the Nuclear and Mitochondrial Genomes. Int. J. Mol. Sci. 2019, 20, 5112. https://doi.org/10.3390/ijms20205112
Alhegaili AS, Ji Y, Sylvius N, Blades MJ, Karbaschi M, Tempest HG, Jones GDD, Cooke MS. Genome-Wide Adductomics Analysis Reveals Heterogeneity in the Induction and Loss of Cyclobutane Thymine Dimers across Both the Nuclear and Mitochondrial Genomes. International Journal of Molecular Sciences. 2019; 20(20):5112. https://doi.org/10.3390/ijms20205112
Chicago/Turabian StyleAlhegaili, Alaa S., Yunhee Ji, Nicolas Sylvius, Matthew J. Blades, Mahsa Karbaschi, Helen G. Tempest, George D. D. Jones, and Marcus S. Cooke. 2019. "Genome-Wide Adductomics Analysis Reveals Heterogeneity in the Induction and Loss of Cyclobutane Thymine Dimers across Both the Nuclear and Mitochondrial Genomes" International Journal of Molecular Sciences 20, no. 20: 5112. https://doi.org/10.3390/ijms20205112