GSM1707342: Cotyledon of seedling biological replicate 3; Glycine max; Bisulfite-Seq
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 compartment was captured from ten-micron sections using a Leica LMD6000.  Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA).  Approximately 300 nanogram of genomic DNA was 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.
GEO Accession
GSM1707342