H the oxidized DNA and the corresponding untreated 500 ng aliquots were bisulfite converted with EZ DNA Methylation-Gold kit (ZymoResearch, catalogue number D5005), and the resultant DNA samples were amplified with the following primers: DROSHA-F, TTTAGTTGGGTGGTTTTATTTG; DROSHA-R, CAACTACTTTTATACCAAC; CDH2-F, GTGGTA GTGGTTGTAATTATATA; CDH2-R, CTTAAAAAATAAATCATTCCTCCC. To increase the specificity of amplification, the first-round PCR products were purified, diluted 1:10,000 and amplified again with the nested primers: DROSHA(n)-F, GTTTTATGTTTT GTGGTAGA; DROSHA(n)-R, ACTTTTATACCAACCTAACA; CDH2(n)-F, ATGAGAAGAGTTATGATAT GGGAAT; CDH2(n)-R, AAAAATAAATCATTCCT CCC. The second-round PCR products were purified and subjected to direct Sanger sequencing (Eurofins MWG Operon, Ebersberg, Germany) [50]. The C/(C+T) ratios from the oxidized samples reflect the 5hmC values, whereas such ratios from corresponding nonoxidized aliquots are equal to the sum of 5mC and 5hmC at the same CpG sites. Hence, the subtraction of the resultant C/(C + T ratios) permits the calculation the 5mC and 5hmC values for each CpG site.Additional materialAdditional file 1: Text S1 (the detailed NGS library preparation protocol), Figures S1 to S5 and Tables S1 to S4. Additional file 2: Tab-delimited text file describing all 5hmC blocks discovered in the epigenomes of fetal livers. Fields 1 to 3 contain genomic coordinates (Hg19) of each 5hmC block. Field 4 indicates the number of 5hmC-positive samples in a given age cohort (that is, those samples that manifest a 5hmC peak in a given genomic interval). Fields 5 and 6 provide some metrics of 5hmC occupancy of given 5hmC blocks; field 5 indicates the sum of 5hmC peak lengths (bp), whereas field 6 shows the sum of NGS reads in 5hmC peaks among all 5hmC-positive samples. Additional file 3: Tab-delimited text file describing all 5hmC PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27906190 blocks discovered in the epigenomes of adult human livers. Fields 1 to 3 contain genomic coordinates (Hg19) of each 5hmC block. Field 4 indicates the number of 5hmC-positive samples in a given age cohort (that is, those samples that manifest a 5hmC peak in a given genomic interval). Fields 5 and 6 provide some metrics of 5hmC occupancy of given 5hmC blocks; field 5 indicates the sum of 5hmC peak lengths (bp), whereas field 6 shows the sum of NGS reads in 5hmC peaks among all 5hmC-positive samples.Received: 24 June 2013 Revised: 30 July 2013 Accepted: 19 August 2013 Published: 19 August 2013 References 1. Ivanov M, Kacevska M, Ingelman-Sundberg M: Epigenomics and interindividual differences in drug response. Clin Pharmacol Ther 2012, 92:727-736. 2. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A: Conversion of 5-methylcytosine to 5hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009, 324:930-935. 3. Kriaucionis S, Heintz N: The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 2009, 324:929-930. 4. Ito S, D’Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y: Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 2010, 466:1129-1133. 5. Iyer LM, Tahiliani M, Rao A, Aravind L: Prediction of novel families of enzymes involved in oxidative and other complex ICG-001 web modifications of bases in nucleic acids. Cell Cycle 2009, 8:1698-1710. 6. Reitman ZJ, Yan H: Isocitrate dehydrogenase 1 and 2 mutations in cancer: alterations at a crossroads.