The identification of differentially methylated genes between hepatocytes and skeletal muscle of wild-type mice
WGBS was performed to examine the different methylation in hepatocytes and muscle. Primary hepatocytes were isolated from 10-week-old male C57BL/6J mice. Hepatocytes from C57BL/6J were purified by density gradient centrifugation with Percoll (Sigma-Aldrich, St. Louis, MO). Isolated hepatocytes were seeded at 5.0 Ã? 104 cells/cm2 to collagen I-coated dish and cultured with DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (NICHIREI BIOSCIENCES, Japan), Penicillin/Streptomycin (10000 U/mL) (Thermo Fisher Scientific, Waltham, MA). After 24 hours, the medium was replaced with serum-free DMEM containing 0.01 nM insulin and 10 nM dexamethasone (Fujifilm-Wako, Japan) and incubated for 16 hours. 100 ng of purified genomic DNA was spiked with 1 ng of unmethylated lambda DNA (Promega), and the mixture was bisulfite converted. Then, the bisulfite-treated DNA was split into two portions and individually served for library preparations in forward and reverse directions. The two libraries of different directions were mixed and served for sequencing. Sequencing was performed with HiSeq X ten at Macrogen Japan (Tokyo, Japan), assigning a lane per sample.