Instrument: Illumina HiSeq 2000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: SINGLE
Construction protocol: Harvested seeds were fixed in 3:1 (v/v) ethanol:acetic acid and embedded in paraffin (Kerk et al., Plant Physiol.132. 27-35 (2003)). Seed parts (seed coat, embryonic cotyledons, and embryonic axis) were captured from ten-micron longitudinal sections using a Leica LMD6000. Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). Approximately 100, 250, and 500 nanogram of genomic DNA isolated from embryonic axis, embryonic cotyledons, and seed coat, respectively, were subjected to library preparation following the methods of Hsieh et al. (Hsieh T-F et al. 2009. Science 324:1451-1454) with modifications. We spiked-in ~ three nanogram of unmethylated lambda DNA (Promega) to serve as a control for complete bisulfite conversion. Adapter-ligated genomic DNA was subjected to two rounds of bisulfite (BS) treatment using the EpiTect kit (Qiagen, Valencia, CA). BS-treated DNA was purified using AMpure XP beads (Beckman) and PCR-amplified for 10 cycles using ExTaq (EpiCentre) DNA polymerase. PCR-amplified DNA fragments were size selected using the AMpure XP beads (Beckman). Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.