title: Whole Genome Bisulfite Sequencing (WGBS) or zebrafish, medaka, and zebrafish-medaka hybrid embryos
design: Whole Genome Bisulfite Sequencing (WGBS) or zebrafish, medaka, and zebrafish-medaka hybrid embryos
library_construction_protocol: Medaka, zebrafish and zebrafish-medaka hybrid embryos were bleached in 0.5% commercial bleach solution, washed, and allowed to develop to the appropriate developmental stage before being dechorionated with 2mg/ml pronase solution. Medaka, zebrafish and zebrafish-medaka hybrid samples were dissolved in homogenization buffer (20 mM Tris pH 8.0, 100 mM NaCl, 15 mM EDTA, 1% SDS) for 3 hours at 550C followed by two rounds of Phenol/Chloroform/Isoamyl Alcohol (25:24:1,PCI) extractions. PCI extraction was performed using an equal volume of PCI to sample and 5 minutes of centrifugation at 13,000 rpm. DNA was then precipitated from the aqueous phase by the addition of 1/10 volume of 3M NaOAc, 20 µg/mL linear acrylamide, 3 volumes of ice-cold ethanol and incubation for 2 hours at -20C. DNA was then pelleted by centrifugation for 30 minutes, washed with 75% ethanol, and resuspended in nuclease-free water. WGBS libraries were prepared from each development stage in biological replicates as follows. 0.5% of unmethylated lambda phage DNA (Promega, Madison, WI, USA) was spiked into each DNA sample before the DNA was sonicated to an average of 300bp. DNA was then purified using AMPure XP beads (Beckman Coulter, Lane Cove, NSW, Australia) and bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo Research, CA, USA) following manufacturer instructions. Bisulfite-converted DNA was then processed to generate WGBS libraries using the Accel-NGS™ Methyl-Seq DNA Library Kit (Swift Biosciences, Ann Arbor, Mi, USA), following manufacturer's instructions.