Analysis of DNA Methylation in replication and cell cycle arrest.

In order to determine the effects of cell cycle and cell cycle arrest on DNA methylation, whole genome bisulfite sequencing was performed on primary fibroblasts sorted based on DNA content and Ki67. Three sets of adult primary dermal fibroblasts were cultured for 4 passages in DMEM with 15% FBS, stained with propidium iodide and anti-Ki67 and sorted into "G0" (2N DNA, Ki67 -), "G1" (2N DNA, Ki67 +) and "G2" (4N DNA, Ki 67 +) using FACS. A subset of passage 4 cells were grown to contact inhibition and maintained for 1 week, then "Ext_G0" cells were isolated. DNA was extracted from all sorted groups, bisulfite converted and sequenced using Illumina HiSeq 2000.