WGBS of Pisaster ochraceus Tube Foot
Specimen was dissected, flash-frozen, and genomic DNA was extracted using a salting-out protocol with an addition of 3M ammonium acetate to precipitate the DNA. DNA was purified using the Zymo DNA Clean & Concentrator 5 kit. The DNA was then fragmented using a Covaris M220 Focused-Ultrasonicator with a target of 200bp, and size range was confirmed using a TapeStation (Agilent). After size selection libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). Bisulfite conversion was performed using the EpiJET Bisulfite Conversion Kit (Thermo Fisher Scientific) with unmethylated lambda phage DNA (NEB Cat#N3013S) included as a conversion control. Bisulfite-converted libraries were PCR amplified using Q5U Hot Start High-Fidelity DNA Polymerase (New England Biolabs) under the manufacturers recommended conditions, with the exception of an annealing temperature of 68C for 8 cycles. The amplified libraries were cleaned up with AMPure XP Beads (Agencourt).