Name: methylC-seq_STL001PO-01b
Instrument: Illumina HiSeq 2000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: SINGLE
Construction protocol: One ug of genomic DNA was extracted from frozen ground tissue using the DNeasy Mini Kit (Qiagen, Valencia, CA) and spiked with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 200 bp, followed by end repair and addition of a 3' A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer's instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 4 cycles of PCR with the following reaction composition: 25 ul 2x KAPA HiFi HotStart Uracil+ReadyMix, 2.5 ul Primer 1, 2.5 ul Primer 2 (50 ul final). The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 4-8 cycles of 98C 15 sec, 60C 30 sec and 72C 4 min, ending with one 72C 10 min step. The reaction products were purified using AMPure XP beads (two rounds). Up to three separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to two independent libraries from the same biological sample.