2.4 | Standard DNA
0% and 100% methylated standard DNA was created to calculate the methylation rate of the samples. High concertation of DNA is needed for the preparation of standard DNA. We used skin and muscle samples from four individuals that were bycatch in gill nets off Mikura Island. The individual number and bycatch years are as follows: #165 and #259 in 2008, #406 in 2013, and an unknown individual in 2005 (Table S1). After, a small piece of skin and muscle sample was cut into smaller pieces, DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN GmbH, Hilden, Germany). The 0% methylated standard DNA was obtained by performing whole genome amplification treatment using the REPLI-g Mini Kit (QIAGEN GmbH, Hilden, Germany). The 100% methylated standard DNA was obtained by fully methylating with CpG methyltransferase (M.SssI; New England Biolabs, Beverly, MA, USA). Each standard DNA was purified using High Pure PCR Product Purification Kit (Roche Molecular Systems, Pleasanton, CA, USA).