DNA methylation was analyzed using bisulfite pyrosequencing. Bisulfite PCR was performed according to the manufacturer’s protocol. One microliter of bisulfite-treated DNA, prepared using an EpiTect Bisulfite Kit (Qiagen, Valencia, CA), was used as a template. The primers used for amplifying the CpG sequences in the given sequence are described in Supplementary Table 1 . After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers (Supplementary Table 1 ). The pyrosequencing reactions were performed using the PyroMark Q24 Advanced (Qiagen) with the 3 HPV-HNSCC cell lines. Primers for the methylation analysis of integration sites in the UPCI:SCC090 line are shown in Supplementary Table 1 .
DNA fragments, including 150 bp of the integrated HPV DNA and 150 bp of the human genome around the boundary, were then analyzed for average methylation to examine the correlation between the methylation pattern of the integrated HPV DNA and that of the human genome.
Lastly, allele-specific DNA methylation of both the integrated HPV genome and adjacent human genome was analyzed as described previously18 (link). For this analysis, the pyrosequencing reactions were performed using the PyroMark Q24 Advanced (Qiagen).
DNA fragments, including 150 bp of the integrated HPV DNA and 150 bp of the human genome around the boundary, were then analyzed for average methylation to examine the correlation between the methylation pattern of the integrated HPV DNA and that of the human genome.
Lastly, allele-specific DNA methylation of both the integrated HPV genome and adjacent human genome was analyzed as described previously18 (link). For this analysis, the pyrosequencing reactions were performed using the PyroMark Q24 Advanced (Qiagen).
