Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells
Five µg of genomic DNA was extracted from frozen cell pellets 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 75-175 bp or 100-400 bp for single-read or paired-read libraries, respectively, 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 as per manufacturer's instructions for genomic DNA library construction. For single-read libraries, adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). For paired-read libraries, adapter-ligated DNA of 275-375 bp (150-250 bp insert) was isolated by 2% agarose gel electrophoresis. 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 4-8 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 µl 10X PfuTurbo reaction buffer, 31 µM dNTPs, 1 µl Primer 1, 1 µl Primer 2 (50 µl final). The thermocycling parameters were: 95 °C 2 min, 98 °C 30 sec, then 4-8 cycles of 98 °C 15 sec, 60 °C 30 sec and 72 °C 4 min, ending with one 72 °C 10 min step. The reaction products were purified using AMPure XP beads.