Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C. Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Dnmt3a (Santa Cruz, sc-20703), H3K4me3 (Millipore, 07-473), H3K27me3 (Millipore, 07-449), total histone H3 (Millipore, 07-690), Ezh2 (Active Motif, 39103) and Suz12 (A kind gift from Dr. Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. J1 ESCs were maintained in standard culture conditions in presense of irradiated mouse embryonic fibroblasts.Įach ChIP-chip assay was performed with about 20 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). To avoid the potential non-specific effect induced by prolonged culturing, only NSCs with limited in vitro culturing (less than 3 weeks) were used in the study. The monolayer cultures were serially passaged every 4-7 days. The NSCs were maintained in serum-free B27 medium in presence of 10 ng/ml FGF-2/bFGF (Peprotech) and 10 ng/ml EGF (Invitrogen). Serial passage (less than 2 passages, or 2 weeks) allowed us to enrich undifferentiated NSCs to high purity (>95% Nestin+/Sox2+). Single cell suspensions were plated at low density (10,000~20,000 cells cm-2) on cell culture dishes coated with poly-L-ornithine (PO)/fibronectin (FN), allowing individual cells to form spatially distinct colonies. Postnatal day 14-21 day mouse lateral ventricle walls were dissected and mechanically dissociated. GEO help: Mouse over screen elements for information.
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