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Fig. 5 | Clinical Epigenetics

Fig. 5

From: Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways in prostate carcinogenesis

Fig. 5

Validation of differentially hydroxymethylated regions (DHMRs) via hMeSeal-qPCR. a Top: IGV Genome Browser representation of absolute hydroxymethylation peaks called in single replicate from hMeSeal-seq data within regions showing hydroxymethylation in both hMeSeal- and hMeDIP-seq and compared to unenriched input control for three representative genes for validation: CUL2, RASGEF1a, and HINT1, and HOXD8 negative control. Peak strength indicated by height of representative peak within replicate or input control (scale shown in square brackets). Colored vertical bars within peaks represent differences between sequenced bases and the hg19 reference genome. Bottom: Blue horizontal bars indicate the presence of introns. Green horizontal bars indicate CGIs. Red boxes indicate the position of the hMeSeal-qPCR amplified segment depicted relative to the overall location of the gene (figure not to scale). Gene diagram adapted from UCSC Genome Browser. b Validation of hydroxymethylation gene enrichment for RWPE-1. Enrichment of hydroxymethylation detected via hMeSeal-qPCR for candidate genes, CUL2, RASGEF1a, and HINT1, within RWPE-1 compared to HOXD8 negative control and normalized relative to 0.03 % input control using the ΔΔCt method (mean values ± standard deviation, n = 3). All genes were compared to negative hMeSeal control reaction performed without UDP-azide-glucose and representing nonspecific binding. c Hydroxymethylated gene enrichment for RWPE-1 versus 22Rv1. Differential detection of genes identified as uniquely hydroxymethylated in RWPE-1, but not 22Rv1, by hMeSeal-seq via hMeSeal-qPCR compared to negative control hMeSeal reaction and normalized relative to 0.03 % input control using the ΔΔCt method (mean values ± standard deviation, n = 3)

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