GSE121005
Genome-wide maps of apurinic sites (AP-sites) and 8-oxoG derived secondary AP-sites
We developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. The experiment was performed in triplicates on HepG2 cells, 30 min after X-ray treatment (6Gy). AP-Seq is an approach that specifically enriches AP-sites via a biotin-labelled aldehyde-reactive probe under pH neutral conditions, which has been well established for the specific detection of AP-sites since its development by Kubo et al. in 1992. Under neutral conditions (pH7), 1h at 37ºC, the probe is highly specific for the aldehydes occurring at AP-sites. After biotin labelling of genomic DNA, DNA is sonicated into ~250 bp fragments, enriched, in vitro repaired (PreCR, NEB), and used for Illumina short read sequencing (125 bp, paired end, HiSeq2000). To assess oxidative damage as the sum of AP-sites and 8-oxoG, we applied recombinant OGG1 in vitro to the extracted DNA (OGG1-AP-seq). Under the conditions chosen, any remaining 8-oxoG is excised in a largely sequence-independent fashion after DNA extraction to result in a set of secondary AP-sites and to a lesser extent the associated beta-elimination product. Overall design: 24 samples, 125bp paired-end illumina sequencing