Genomeplex

DNA was harvested from cells harboring a single integrated BrdU incorporation vector [19] (link) and replicated DNA was immunoprecipitated as described [20] (link) with the following modifications: Lysis buffer contained 100 mM Tris-HCl pH 8.0 and 20% glycerol, cell lysate was sonicated using a Bioruptor sonicator bath (Diagenode), and BrdU immunoprecipitation was performed using Dynabeads Protein G (Novex). Input (G1 phase) and immunoprecipitated S-phase DNA was amplified using whole genome amplification (GenomePlex, Sigma-Aldrich).

Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested with nocodazole (15 µg/ml) for 2 hours at 30°C. Samples were cross-linked with 1% formaldehyde for 20 min at room temperature, and cross-lining was stopped with 0.125M glycine. Chromatin was sonicated using a Bioruptor sonicator bath (Diagenode), to an average fragment size of 250 bp. Soluble chromatin was immunoprecipitated as previously described [18] (link) using antibodies against Flag (Sigma). Immunoprecipitation efficiency was checked by Western blot. Immunoprecipitated and input DNA were amplified using whole-genome amplification (Genome-Plex, Sigma-Aldrich).

Array-based DNA methylation profiling was accomplished by using the Illumina GoldenGate Cancer Panel I methylation bead array to simultaneously interrogate 1505 CpG loci associated with 807 cancer-related genes. We previously determined that this array showed high reproducibility; results obtained in FFPE tissues were highly correlated with those from matched non-FFPE samples (r =0.97), and published tumor-specific methylation profiles were detectable when DNA specimens contained at least 70% tumor cells [21 (link)].
Bead arrays were run in the Mammalian Genotyping Core laboratory at UNC at Chapel Hill. The Illumina GoldenGate methylation assay was performed as described previously [22 (link)] and imaged by using the BeadArray Scanner. Methylation status of the interrogated CpG sites was determined by comparing the ratio of the fluorescent signal from the methylated allele with the sum from the fluorescent signals of both methylated and unmethylated alleles. Controls for methylation status used on each bead array included the Zymo Universal Methylated DNA Standard as the positive, fully-methylated control, and a GenomePlex (Sigma-Aldrich, St. Louis, MO, USA) whole genome amplified DNA used as the negative, unmethylated control. Array data have been deposited in Gene Expression Omnibus under accession number GSE51557.

Methylated DNA isolation assay (MeDIA)-assisted CpG microarray analysis was performed as described previously with slight modification [31 (link)]. Briefly, 1.0 μg sonic-fragmented genomic DNA from the test and control tissues, respectively, was incubated with 2 μg recombinant MBD2bt protein for 4 h at 4 °C on a rocking platform. The enriched, methylated DNA was amplified using a whole-genome amplification kit (GenomePlex®, Sigma, USA) according to the manufacturer’s protocol. The amplified DNA in the control and test samples was labeled with Cy5 and Cy3, respectively. The labeled samples were purified using a PCR purification kit (Qiagen, USA) and then co-hybridized to human promoter 1 M microarrays (Agilent, Santa Clara, CA, USA) according to manufacturer’s instructions.