Genome-wide connection between DNA methylation and DNA replication timing

DNA methylation and DNA replication timing were examined across a variety of human tissues and cell lines, applying microarray-based techniques. The analyses revealed that late-replicating DNA was demethylated compared to the methylation of early-replicating regions. DNA methylation: Epstein-Barr-Virus (EBV) transformed B-lymphocyte cell lines GM10849, GM12089, GM12092, GM12093, and GM08714 (ICF) (http://ccr.coriell.org/nigms) were cultured in RPMI-1640 supplemented with 15% FCS (Sigma) at 37oC and 5% CO2. Normal BJ foreskin fibroblasts (NHF cells) at different PDs (36)(34) were cultured in 4:1 DMEM : M-199 supplemented with 15% FCS (Sigma) at 37oC and 5% CO2. Replication timing: EBV-transformed female B-lymphocyte cell lines GM12092 and GM12093 were cultured as above, harvested from logarithmic growth cultures, washed once in ice-cold phosphate-buffered saline, immediately fixed in 70-85-95% ethanol at -20oC, stained with propidium iodide, and sorted into G1 and early-S fractions using a MoFlo cell sorter.