Additional file 2.

Optimization of bisulfite sequencing PCR. MeDIP validation (Figures S1 and S2). Bisulfite sequencing of glutathione S-transferase P1 gene (gstp1) (Figure S1) showed complete un-methylation of the CpG dinucleotides. Bisulfite sequencing of the no-tail (ntla) gene showed complete methylation of the CpG dinucleotides (Figure S2). (Sequencing was performed on 4 independent samples; two tumor samples and two control samples, arrows: cytosine located at CpG dinucleotide site. Red peak: T, Blue peak: C) Validation of bisulfite sequencing PCR (Figure S3). Glutathione S-transferase P1 gene is un-methylated in both tumor and control. Treatment with CpG methyltransferase (Sss I) in the presence of S-adenosylmethionine (SAM) results in methylation of all CpG dinucleotides and protection against conversion of 5-mC to U after treatment with bisulfite. Successful bisulfite treatment and sequencing of the positive control was established by observing a single C peak at the CpG dinucleotide positions. (Two independent tumor samples, two independent control samples). 1 = SAM positive control, 2 = control, 3 = control, 4 = tumor, 5 = tumor. Arrows: cytosine located at CpG dinucleotide site.

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Mirbahai et al. BMC Genomics 2011 12:3   doi:10.1186/1471-2164-12-3