ChIP-seq analysis of Foxa2 binding sites in midbrain dopaminergic progenitors
ChIP-seq analysis of Foxa2 binding sites in midbrain dopaminergic progenitors
NestinLmx1a-transfected E14.1 ES cells were propagated on gelatinized culture dishes in DMEM (Invitrogen) supplemented with 2000 U/ml LIF (Chemicon), 10% KSR, 2% FCS, 0.1 mM nonessential amino acids, 1 mM pyruvate (Invitrogen), and 0.1 _M _2-mercaptoethanol (Sigma). For in vitro differentiation, 1.2 _ 106 ES cells were nucleofected with expression vectors using the mouse ES nucleoporator kit (AMAXA), replated on gelatinized 24-well plates (15,000 cells/well), and incubated in ES medium for 12D15 hr. ES cells then were differentiated to mDA cells in N2B27 medium supplemented with 20 ng/ml bFGF (Invitrogen), 100 ng/ml FGF8, and 1.7 nM Shh (R&D systems) for 5 days. These in vitro generated mDA progenitors robustly expressed Foxa2 at d5 and differentiated further into Nurr1+/TH+ mature mDA neurons by d8. Cells were cross-linked in 1% formaldehyde for 10 min, and cross-linking was quenched by adding glycine to a final concentration of 0.125 M for 5 min under constant rotation. After washing in PBS to remove excess formaldehyde and glycine, 2 _ 107 fixed cells were homogenized in 300 _L of cold whole-cell lysis buffer (10 mM TrisDHCl at pH 8.0, 10 mM NaCl, 3 mM MgCl2, 1% NP-40, 1% SDS) and protease inhibitors. Lysates were incubated on ice for 10 min and sonicated with a Diagenode Bioruptor (30-s on/off pulses for 10 min, on high setting). Debris was removed by centrifugation at 13,000 x g for 10 min, and the supernatant was collected and snap frozen in liquid nitrogen. As input, 10 _L of sonicated chromatin was incubated in PBS with 200 mM NaCl overnight at 65  C, treated with proteinase K and purified using the QIAquick PCR Purification Kit (Qiagen). Chromatin concentration was determined with a NanoDrop 3.1.0 (Agilent Technologies, Santa Clara, CA, USA). 10 ug of chromatin per sample was immunoprecipitated with 2 _g of rabbit anti-Foxa2 (Besnard et al. Gene Expr. Patterns 5: 193D208) or normal rabbit anti-IgG antibody (Millipore #12-370). Immunoprecipitated DNA was analyzed by real-time quantitative PCR (qPCR) prior to library construction. Extraction was carried out according to manufacturer's specifications.