Briefly, 10 μg of whole blood genomic DNA was sonicated by using a 2 mm probe at amplitude 40 for 30 cycles with 15 seconds on and off, to obtain fragment sizes of 100–800 base pairs. About 3 μg of sheared DNA (INPUT) was immunoprecipitated overnight with anti-5-mathyl cytosine antibody (Diagenode, Belgium) along with positive and negative control DNA provided in the Diagenode kit. Enriched methylated fragments were purified by standard phenol-chloroform-ethanol precipitation [26 (link)]. The quality analysis of enriched immunoprecipitated (IP) methylated fragments was evaluated by real time PCR with positive, negative, GAPDH and AX1 primers (Diagenode, Belgium) [27 (link)]. INPUT and IP were differentially labeled with cyanine3 and cyanine5 (Amersham Biosciences, USA) by indirect method using Bio-prime Array CGH kit (Invitrogen, USA) [28 (link),29 (link)]. Equal concentrations of labeled INPUT and IP DNA were co-hybridized onto the Agilent 244K Human CpG Island, high density oligonucleotide array at 65°C for 40 hours with the continuous rotation at 18 rpm as per the Agilent MeDIP protocol version 1.1 (Agilent Technology, USA). The slide was washed using wash buffers (Agilent Technologies, Santa Clara, CA, USA), dried and scanned using G2505B DNA microarray scanner (Agilent Technology, USA) with Sure Scan High resolution technology (Additional file 1 ).
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