title: Genomic Busulfite seq for Lampyre (Petromyzon marinus) Heart
design: Lamprey specimens were sampled from the Great Lakes in Michigan U.S.A. Genomic DNA of heart was purified from a male lamprey using DNeasy blood & tissue kit (QIAGEN). Input gDNA (5μg) is fragmented by Covaris shearing. The fragments are blunt ended and phosphorylated, and a single ʹAʹ nucleotide is added to the 3ʹ ends of the fragments in preparation for ligation to a methylated adapter that has a single‐base ʹTʹ overhang. The ligation products are purified and size selected by agarose gel electrophoresis. Size selected DNA is bisulfite treated and purified. The treated DNA is PCR‐amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantitated prior to cluster generation. The final concentration of the WGBS library is lower than 10 nM, therefore the subsequent cluster generation protocol must be modified as follows:  1.Add 1 μl 0.2N NaOH to 1 μl of the original WGBS library.   2.Let the tube stand for 5 minutes at room temperature.  3.Add 120 μl Hybridization buffer to the tube to dilute the library.  4.Place the tube on ice until till it is ready for cluster generation.