Dna preparation kit and protocol

EpiQuest libraries were prepared according to Zymo Research Protocol (Irvin, CA). 200–500 ng of genomic DNA were digested with NEB 60 units of TaqI and 30 units of MspI (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 Zymo Research (ZR) DNA Clean and Concentrator™-5 kit (Irvin, CA). Ligation to preannealed adapters containing 5′-methyl-cytosine was performed using Illumina's DNA preparation kit and protocol (San Diego, CA). Purified adaptor ligated fragments were bisulfite-treated using the EZDNA Methylation-Direct™ Kit (Irvin, CA). 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 were excised. Library material was recovered from the gel (Zymoclean™ Gel DNA Recovery Kit, Irvin, CA, USA) and sequenced on an Illumina HiSeq Genome Analyzer (San Diego, CA).

DNA samples were shipped on ice to Zymo Research (Irvin, CA) for EpiQuest library preparation and genome-wide DNA methylation analysis by reduced representation bisulfite sequencing (RRBS). Briefly, 200–500 ng of genomic DNA were digested first with TaqI followed by digestion with MspI (Ipswich, MA, USA). Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled-in and 3′-terminal-A extended, then extracted by Zymo Research DNA Clean and Concentrator-5 kit (Irvin, CA). Ligation to pre-annealed adapters containing 5′-methyl-cytosine was performed using Illumina’s DNA preparation kit and protocol (San Diego, CA). Purified adaptor-ligated fragments were bisulfite-treated using the EZ DNA Methylation-Direct Kit (Irvin, CA). 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 were excised. Library material was recovered from the gel (Zymoclean Gel DNARecovery Kit, Irvin, CA, USA) and sequenced on an Illumina HiSeq Genome Analyzer (San Diego, CA).