GSM7439093
Eight to ten days after pollination (DAP) developing F1 seeds (linear to bending cotyledon stage) were immersed in dissection solution (filter-sterilized 0.3 M sorbitol and 5 mM pH 5.7 MES) on sticky tape and dissected by hand under a stereo-microscope using fine forceps (Fine Science Tools, Inox Dumont #5) and insect mounting pins. The seed coat was discarded, and debris removed by washing collected embryos or endosperm five to six times with dissection solution under the microscope. Genomic DNA was isolated using CTAB method. Bisulfite sequencing libraries for Illumina sequencing were constructed as in (Ibarra et al 2012) with minor modifications. In brief, about 50 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer's instructions for gDNA library construction (Illumina).  Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer's instructions. The bisulfite-converted library was split between two 50 ul reactions and PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 μl of 10X ExTaq reaction buffer, 25 μM dNTPs, 1 μl Primer 1.1 and 1 μl multiplexed indexing primer.  PCR reactions were carried out as follows: 95ºC for 3 minutes, then 14-16 cycles of 95 ºC 30 s, 65 ºC 30 s and 72 ºC 60 s. Enriched libraries were purified twice with AMPure beads (Beckman Coulter) prior to quantification with the Qubit fluorometer (Thermo Scientific) and quality assessment using the DNA Bioanalyzer high sensitivity DNA assay (Agilent).  Sequencing on either the Illumina HiSeq 2000/2500 or HiSeq 4000 platforms was performed at the Vincent J. Coates Genomic Sequencing Laboratory at UC Berkeley.