(5) 501 to 1,000 bps and (6) 1,001 to 1,500 bps. Average DNA range (IQR), whiskers the last data point within ±1.5 x IQR, bars methylation was calculated for probes in three groups based on the median and dots outliers. CpG island density within each of the six TSS bin. Error bars give the standard deviation of this average between somatic tissue Disclosure of Potential Conflicts of Interest samples. Given that we found no difference in DNA methyla- No potential conflicts of interest were disclosed. tion based on direction to TSS, probes were not separated into or upstream/downstream in later TSS analyses. Acknowledgments Global DNA Methylation. Total 5-mC content of each sample was measured using the MethylFlash Methylated DNA Quantification Kit (Colorimetric) (Epigentek Group Inc.) fol- lowing the manufacturer’s protocol. As our results were variable and correlation of technical replicates was not significant, these data were not included in this publication. Statistical analyses. Comparison of methylation between tissue groups, gestational ages and RE evolutionary age was performed using the non-parametric ANOVA, Kruskal-Wallis test, followed by Dunn’s multiple comparison tests. Averages of DNA methylation are stated for tissue groups ± standard devia- tion. Spearman’s rank order correlation (r) was used to compare DNA methylation of different ReDS within each tissue group and within an individual, followed by Benjamini-Hochberg cor- rection for multiple comparisons. A chi-square test was used to compare the observed vs. expected number of probes in each of the TSS bins. Statistics were performed in GraphPad Prism Supplemental Material 5 (GraphPad Software, Inc.). G©raphs 2we0rep1rod2uceLdianRnadnd eSsuppBlemieontaslmcatieerialnsmcayebe.downloaded here: GraphPad Prism 5. For box plots, boxes represent the interquartile www.landesbioscience.com/journals/epigenetics/article/20221 The authors would like to thank Ruby Jiang for technical assis- tance, Courtney Hanna and Dr. Kirsten Hogg for their con- structive comments, Lucia Lam and Sarah Neumann for aid in microarray processing and analysis, Dr. Angela Devlin for use of the Biotage PyroMarkTM MD machine, Dr. Dixie Mager for useful conversations and Katarzyna Stepien for her figure illus- trations. This work was supported by funds from the BC Clinical Genomics Network, a grant from the Canadian Institute for Health Research to W.P.R., and a training fellowship from the Child and Family Research Institute to E.M.P. M.S.K. is a Scholar of the Michael Smith Foundation for Health Research and the Canadian Institute for Advanced Research. 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