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).