Genomic DNA was extracted from 3 dnmt1+/+ and 3 dnmt1-/- medaka at 18 dpf using DNeasy blood and tissue kit (Qiagen). 150ng of DNA was used for the bisulfite reaction using the EZ DNA methylation kit (Zymo Research) and the bisulfite-treated DNA was recovered in 15ml of elution buffer. 1ml of bisulfite-treated DNA solution was used for 30 cycles of PCR amplification with the indicated bisulfite primers in a reaction volume of 20ml; 1ml of the first PCR reaction was used for a nested PCR reaction with P5 and P7 adaptor-attached bisulfite primers. After the nested PCR reactions, amplicons were purified using PCR purification kit (Qiagen) and qualified and quantified by gel electrophoresis, the High Sensitivity DNA kit (Agilent) for the Agilent Bioanalyzer and the Qubit dsDNA HS assay kit (Invitrogen). The eight amplicons derived from the same original source were mixed at 1ng/ml each and 15ml of the mixture were amplified using P5 and P7 primers attached to barcodes using NEBNext DNA library prep kit (New England Biolabs), following the manufacturer«s protocol. The amplicons were purified using AMPure XP (Beckman Coulter) and qualified and quantified using Bioanalyzer and Qubit dsDNA HS assay kit. Finally, all amplicons were mixed at 1ng/ml each and 5 ml of mixed amplicons were used for sequencing in paired-end 150bp mode on 1 lane of MiSeq Sequencing System (Illlumina).
GEO Accession
GSM2607704