5-hmC contained DNA were enriched as described(Robertson et al., 2012) with modifications. Fragmented DNA was then subjected to end repair with the end repair enzyme mix (NEB), dA tailing with the Klenow 3´-5´ exo- (NEB) and ligation of multiplexing adapters (Illumina) using T4 DNA ligase (NEB). Adapter-ligated DNA of 280-500bp was purified by 2％ agarose gel electrophoresis, and then 5hmC was converted to β-glucosyl-5-hmC (β-glu-5-hmC) by T4 β-glucosyltransferase. β-glu-5-hmC contained DNA fragment was further pulled down by J-binding protein 1 (JBP1)-coated magnetic beads from Quest 5-hmC™ DNA Enrichment Kit (zymo research). Finally, enriched DNA fragments that have adapters on both ends were amplified using PCR to create the final sequencing library and sequenced by Illumina HiSeq 2000. A quantity of fragmented genomic DNA was set aside as input DNA. The efficiency of precipitation was checked by kit included positive control fragments.