Genomeplex complete whole genome amplification kit

Methylated DNA fragments from one sonication aliquot per sample were enriched for methylcytosine using methyl-CpG binding domain-based capture (MBD-Cap) with the EpiMark Methylated DNA Enrichment Kit (New England BioLabs, Ipswich, Massachusetts). The EpiMark kit uses a fusion protein (MBD-Fc) containing the highly conserved methyl binding domain (MBD) of human MBD2 protein fused to the Fc tail of human IgG. MBD-Fc proteins and paramagnetic protein A beads were incubated for 15 minutes at room temperature, allowing Fc domains to couple with protein A beads, and washed twice with wash buffer. Fragmented genomic DNA was added to MBD-Fc/bead mixture, rotated at room temperature for 20 minutes, and washed three times with wash buffer to discard unbound DNA. Captured methylated DNA was eluted with 100 μl of DNase-free water during a 15-minute incubation at 65 ºC. Residual unmethylated DNA fractions were reserved for downstream enrichment testing via qPCR. In order to obtain sufficient DNA for microarray hybridization, 10 ng of captured methylated DNA was amplified using GenomePlex Complete Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, Missouri).

Input and anti-γH2AX immunoprecipitated chromatin was uncross-linked and genomic DNA was purified using DNA spin columns (Qiaquick, Qiagen). Samples were amplified using the GenomePlex Complete Whole Genome Amplification kit (Sigma) without further fragmentation, to obtain sufficient quantities of DNA (>2 μg) for labelling and microarray hybridization. Input and confluent or scratched samples were co-hybridized to human 385 K RefSeq Promoter arrays (Roche Nimblegen), according to the manufacturer’s protocol, and analysed using DEVA v1.2.1 software. Peaks were mapped to human genome build GRCh36.1 (NCBI) and verified in build GRCh37.3. The expected number of DSBs for a given genomic region was determined by dividing the number of promoters in the region by the total number of promoters represented on the chip, then multiplying by the total number of peaks observed at a false discovery rate of 0.20. The ratio of observed-to-expected number of peaks was then plotted by chromosome and analysed using Fisher’s exact test.

MSI testing was performed on all samples subjected to whole-exome sequencing using the marker set employed by TCGA^{3 (link)}. DNA was whole-genome amplified using the GenomePlex Complete Whole Genome Amplification kit (Sigma Aldrich). The probe set consisted of BAT25, BAT26, BAT40, TGFBRII, D2S123, D5S346 and D17S250. No markers were positive in the normal controls (blood). None of the patients in this study were diagnosed with hereditary nonpolyposis colorectal cancer (HNPCC).

DNA was isolated from whole blood or from blood spot cards using either the QIAamp DNA Mini and Micro Kits (Qiagen, Hilden, Germany), the Extract-N-Amp™ Blood PCR Kit (Sigma-Aldrich, St. Louis, MO, USA) or salting out applying a manual protocol or a semi-automated protocol based on the Autopure System (Qiagen, Hilden, Germany). For 336 of the samples, DNA was amplified using the GenomePlex® Complete Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO, USA) prior to genotyping.
Genotyping of the APOE variants rs429358 and rs7412 and the FOXO3A variants rs7762395 and rs479744 were primarily carried out using predesigned TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) with genotyping efficiencies of between 96,8% and 99,7%. For 641 of the 1905 Birth Cohort Study participants, genotyping of the FOXO3A variants were performed as part of a previous study using the Illumina GoldenGate Technology (Illumina Inc, San Diego, CA, USA) as described by Soerensen et al. 2012 (link) (Soerensen et al. 2012 (link)).