EpiQuest libraries were prepared according to manufacturer’s protocol [Zymo
Research (ZR), Irvine, CA, USA] and as previously described.16 (link) Genomic DNA (200–500 ng) was digested with 60 units of
TaqI and 30 units of MspI
(NEB, Ipswich, MA, USA) sequentially. Size-selected
TaqI-MspI fragments (40–120 bp and
120–350 bp) were filled-in and 3′-terminal-A extended, extracted with a ZR DNA
Clean and Concentrator™-5 kit. Ligation to pre-annealed adapters
containing 5′-methyl-cytosine was performed using the Illumina’s DNA preparation
kit and protocol (Illumina, San Diego, CA, USA). Purified adaptor ligated
fragments were bisulfite-treated using the EZ DNA Methylation-Direct™Kit (ZR). Preparative-scale PCR was performed. DNA Clean and
Concentrator™ -purified PCR products were subjected to a final
size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices
containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size was
excised. Library material was recovered from the gel (Zymoclean™ Gel
DNA Recovery Kit, ZR) and sequence on an Illumina HiSeq Genome Analyzer.
Research (ZR), Irvine, CA, USA] and as previously described.16 (link) Genomic DNA (200–500 ng) was digested with 60 units of
TaqI and 30 units of MspI
(NEB, Ipswich, MA, USA) sequentially. Size-selected
TaqI-MspI fragments (40–120 bp and
120–350 bp) were filled-in and 3′-terminal-A extended, extracted with a ZR DNA
Clean and Concentrator™-5 kit. Ligation to pre-annealed adapters
containing 5′-methyl-cytosine was performed using the Illumina’s DNA preparation
kit and protocol (Illumina, San Diego, CA, USA). Purified adaptor ligated
fragments were bisulfite-treated using the EZ DNA Methylation-Direct™Kit (ZR). Preparative-scale PCR was performed. DNA Clean and
Concentrator™ -purified PCR products were subjected to a final
size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices
containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size was
excised. Library material was recovered from the gel (Zymoclean™ Gel
DNA Recovery Kit, ZR) and sequence on an Illumina HiSeq Genome Analyzer.
