Verification of MeDIP microarray results.
Bisulfite sequencing (BS), COBRA analysis and real-time qPCR were used to confirm KSHV DNA methylation profiles at select loci. A: Three loci for which our MeDIP analysis had indicated profound methylation, and three loci which were predicted to be unmethylated were analyzed by bisulfite sequencing of BCBL1-derived DNA. The global BCBL1 MeDIP methylation profile and the location of sequenced regions are shown for reference at the top. The results of the bisulfite sequencing are shown underneath, where closed and open circles indicate methylated and unmethylated CpG motifs, respectively. The nucleotide positions indicate the position of the first and the last CpG motifs within the KSHV reference genome (NC_009333). B–E: Confirmation of DNA methylation profiles at the genomic ORF23 locus in PEL cells (HBL6, AP3 and BCBL1), long-term in vitro infected endothelial SLK cells (SLKp), SLK cultures 5 days after de novo infection (SLK-5dpi), in vitro methylated or unmethylated KSHV bacmids (BacM and Bac, respectively), and virion DNA. The methylation profiles of the samples investigated by MeDIP are shown in B. Black lines indicate the regions for which COBRA analysis and bisulfite sequencing of genomic DNA, or real-time qPCR of MeDIP samples were performed. C: Real-time qPCR was performed to quantify immunoprecipitated DNA from three independent MeDIP experiments. Values were calculated as percent of the input and were normalized to an internal control consisting of methylated plasmid DNA (pCR2.1) spiked into each sample prior to MeDIP. D+E: the region indicated in B was PCR-amplified from bisulfite converted DNA and subjected to a COBRA assay (D) or bisulfite sequencing (E). Cleavage of bisulfite converted DNA at the TaqI sites indicated by arrows requires methylation of the corresponding CpG motif. The CpG profiles as shown in E were determined by bulk sequencing reactions except for the samples labeled SLKp #1 and #2, which represent two individual clones from the SLKp line.