LAUSR

ARPE-19 cells, SW1353 cells and Jurkat cells were purchased from the Cell Bank (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). ARPE-19 and SW1353 cells were cultured with DMEM/F-12 medium (Biological Industries, Biet Haemek, Israel), and Jurkat was cultured with RPMI 1640 medium (Biological Industries), both containing 10% fetal bovine serum (Gibco BRL/Invitrogen, Carlsbad, USA), 100 μg/ml streptomycin, and 100 IU/ml penicillin, and incubated at 37oC and 5% CO2. After 48 h during which the cells roughly doubled in number, half of the cells were collected and their DNA was extracted, while the remaining cells continued to culture. Following DNA replication in S phase, it is well known that there is a time delay before DNA methylation of the newly synthesized DNA, resulting in a significantly lower level of genomic DNA methylation in the S phase compared with other phases (G1/G0 and G2/M) [1]. Thus, to avoid methylation heterogeneity caused by some cells in S phase, we cultured cells to the 20 and 30 generations, and then conducted serum starvation for 24 h to synchronize the cells to the G0 phase before collecting the cells. The ARPE-19 and SW1353 cells were then desorbed from the culture dish by digestions with 0.25% Trypsin-EDTA (the Jurkat cells are suspended cells) (Thermo Scientific, Waltham, USA) and collected for DNA extraction.