One microgram of genomic DNA was spiked with 5 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, Wisconsin, USA). The DNA was fragmented with a Covaris S2 (Covaris,Woburn, Massachusetts, USA) to 150–200 bp, followed by end repair and addition of a 3’ adenine base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, California, USA) were ligated to the sonicated DNA at 16 0C for 16 h with T4 DNA ligase (New England Biolabs). Adaptor-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, Massachusetts, USA). Adaptor-ligated DNA (<= 450 ng) was subjected to sodium bisulphite conversion using the MethylCode kit (Life Technologies, Carlsbad, California, USA) as per the manufacturer’s instructions. The bisulphite-converted, adaptor-ligated DNA molecules were enriched by eight cycles of PCR with the following reaction composition: 25 ul of Kapa HiFi Hotstart Uracil1 Readymix (Kapa Biosystems, Woburn, Massachusetts,USA) and 5 ul TruSeq PCR Primer Mix (Illumina) (50 ul final). The thermocycling parameters were: 95 0C 2 min, 98 0C 30 s, then four cycles of 98 0C 15 s, 60 0C 30 s and 72 0C 4 min, ending with one 72 0C 10-min step. The reaction products were purified using AMPure XP beads. Two separate PCR reactions were performed on subsets of the adaptor-ligated, bisulphite-converted DNA, yielding up to two independent libraries from the same biological sample.
GEO Accession
GSM2091089