DNA methylation analysis of mouse embryonic fibroblasts (MEFs) and Thymidine treatment-induced MEFs (induced cells).

Investigation of DNA methlation changing in thymidine treatment-induced MEFs. Mouse embryonic fibroblasts (MEFs) were obtained at E14.5 embryos and supplied with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 15% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Potential hematopoietic progenitor cells (CD45+CD41+CD31+c-Kit+) were negatively selected with Biotin labeled anti-CD45, anti-CD31, anti-CD41 and anti-c-Kit antibodies by magnetic beads. Hereafter, treated MEFs are referred to as 4neg MEFs for initiation of induction. 4neg MEFs were seeded on mitomycin (MMC)-treated MEFs as feeder cells and cultured in DMEM with 15% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 μM thymidine as induction medium for 2 days followed with analysis DNA was purified from MEFs in the absence or presence of thymidine for 2 days. DNA samples were sheared into 200-1000 bp fragments by Bioruptor sonicator (Diagenode). Sonicated genomic DNA was immunoprecipitated by anti-5-methylcytosine antibody (Diagenode) followed by purification using Qiagen MinElute columns. The purified DNA was quantified using a nanodrop ND-1000, labeled by the NimbleGen Dual-Color DNA labeling Kit and array hybridized according to the manufacturer’s protocol (Nimblegene Systems, Inc., Madison, WI, USA).