title: GSM2417691: Early maturation-stage seed coat biological replicate 1; Glycine max; Bisulfite-Seq
library_construction_protocol: Collected tissues were quickly frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Genomic DNA  was isolated from the powder using the DNEASY Plant Mini kit (Qiagen, Valencia, CA) according to manufacturer’s instructions.Approximately one microgram 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. Two libraries were constructed from genomic DNA isolated from seed coat. One library was spiked in ~ three nanogram of unmethylated lambda DNA as all other libraries; the other library was spiked in ~ three nanogram of methylated lambda DNA to serve as a control for over conversion (i.e. conversion of methylated cytosine to uracil) during the bisulfite conversion step. The methylated lambda DNA was generated by treating unmethylated lambda DNA with CpG Methyltransferase (New England BioLabs). 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.
attributes.tag: GEO Accession
attributes.value: GSM2417691