Nimblegen array hybridization kit

DNA samples of three MZ twin sets (#1/2, 9/52, and 24/57; see Table 1) were sonicated and then immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine (Roche NimbleGen, Madison, WI, USA). DNA fragments were converted into PCR-amplifiable OmniPlex™ Library molecules flanked by universal primer sites and the library amplified by PCR using universal primers and a limited number of cycles. Immunoprecipitated and reference DNA were tagged, respectively, with cyanine-5 (Cy5) and cyanine-3 (Cy3)-labeled random 9-mers and then hybridized by the NimbleGen Array Hybridization Kit (Roche NimbleGen, Madison, WI, USA).
A four-plex array was custom-designed to include 998 X chromosome and 18,086 randomly selected autosomal chromosome promoter sites (Roche NimbleGen, Madison, WI, USA) and samples analyzed following the manufacturers protocols. First, Nimblescan software (Roche NimbleGen, Madison, WI, USA) was utilized for DNA methylation data analysis using a threshold p-value of 0.05 equivalent to 1.31 based on the Gaussian distribution of data. Second, exclusive elements corresponding to specific microarray probes were identified in affected and healthy subjects and peaks found only in either group were selected for further analysis. Third, elements of interest were inserted into the UCSC Genome Browser (GRCh36/hg19) to identify corresponding genes.

DNA samples from PBC patients (n = 10) and controls (n = 10) were sonicated to generate fragments between 200 and 1000 base pairs (bp), and then immunoprecipitated with a murine monoclonal antibody that specifically recognizes 5-methylcytidine (Diagenode, Liège, Belgium). The immunoprecipitated DNA fragments were recovered using anti-mouse IgG magnetic beads. The MeDIP-enriched DNA was amplified by PCR using universal primers and a limited number of cycles (GenomePlex® Complete Whole Genome Amplification kit). The amplified DNA samples were then purified with QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). One microgram of immunoprecipitated and reference DNA was tagged respectively with Cyanine-5 (Cy5) and Cyanine-3 (Cy3) labeled random 9-mers and then hybridized by the NimbleGen Array Hybridization Kit (Roche NimbleGen, Madison, WI, USA). The labeled DNA was then purified by isopropanol/ethanol precipitation. For array hybridization, Roche NimbleGen’s Human Promoter plus CpG Island array was used, a 3 × 720k format array design containing 27,728 CpG islands and all well-characterized promoter regions (from about −2440 to +610 bp of the TSSs) totally covered by ~720,000 probes.