Sualization software, Cytoscape (v3.1.0) [57]. Groups of related pathways (nodes) were identified using the MCODE cluster app and were then manually summarized (circled and labeled in figures) [58]. Additionally, GREAT results were organized into bar graphs according to the recommended statistic of -log10 (raw hypergeometric p value) for ranking. Additionally, NGS (raw peaks) and microarray (raw gene expression) data were visualized using Circos software [59].Locus-specific DNA hydroxymethylation detection using hMeSeal-qPCRDNA hydroxymethylation levels were analyzed by RTqPCR using the 7500 Real-Time PCR Instrument (Applied Biosystems). Hydroxymethylation levels were calculated using 2-Ct method, relative to 2.7 of input genomic DNA, and normalized to 0.027 of input genomic DNA. A negative control reaction was performed according to the Hydroxymethyl get DS5565 Collector protocol (Active Motif), where UDP-azide-glucose was excluded from the glucosylation reaction. In addition, 2.5 g of sheared genomic DNA was subjected to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 each glucosylation reaction, for both sample and negative control reactions. The PCR assays comprised of 5 l of PerfeCTa?SYBR?Green FastMix? Low ROX (Quanta Biosciences), 2 l of bound DNA elution (hydroxymethylated or negative control), and 5 M of each primer, with a total volume of 10 l. The 2.7 and 0.027 of input PCR assays were completed using the identical conditions, and input DNA stocks were prepared at 33.3 and 0.33 ng/l, respectively. All PCR assays included a non-template control, using UltraPure Distilled Water (Invitrogen) as the template. The PCR conditions were as follows: 30 s at 95 , 40 cycles of denaturation for 5 s at 95 , and annealing for 30 s at 60 . Primers were designed using the specifications recommended by Hydroxymethyl Collector protocol (Active Motif) and targeted differentially hydroxymethylated regions (DHMRs) identified by both hMeSeal-seq and hMeDIP-seq. The sequences of primers amplifying: RASGEF1a are 5-GCA TGT TCC TTG AAC TGT GA-3 (forward), 5-TCA CAC CCT TCC CAA CAC TA-3(reverse); HINT1 are 5- CAT ATC CAA ATT GCC AGG AT-3 (forward), 5- GCT GAC TTT GCT TTC AGA CC-3 (reverse); CUL2 are 5-GGG GTG CAA TAT CTC ACT GT-3 (forward), 5-GCT TGG AGA AGA CAC ACA AA-3 (reverse); and HOXD8 are 5-AAC TTG CGG TCG TCT GCC CT-3 (forward), 5-ACA GAA ACG TTC TGA GGC GGG AAA-3 (reverse). PCR reactions were performed across three technical replicates.Accession numbersThe data sets supporting the results of this article are available in the Gene Expression Omnibus (GEO) repository, under accession number GSE74464.Additional fileAdditional file 1: Co-occurrence of methylation and hydroxymethylation in pathway regulation. This file lists all pathways found to be co-occurrent between 5mC- and 5hmC-enriched regions within each cell line, providing the genomic features and gene lists associated with enrichment for each mark.Following hMeSeal technique performed on a separate biological replicate from hMeSeal-Seq (previously described),Competing interests The authors declare that they have no competing interests.Kamdar et al. Clinical Epigenetics (2016) 8:Page 16 ofAuthors’ contributions SNK carried out the pathway analysis, performed hMeSeal-qPCR and data interpretation, and drafted the manuscript. LH performed 5mC and 5hmC enrichment, submitted sequencing samples, and helped to draft the manuscript. SNK and LH are considered as equal co-first authors in this study. RH and GB helped with pathway analysis and inte.