ChIP-qPCR Analysis of FOXO3a Binding in Bone Marrow

ChIP was carried out in WT total bone marrow TER119⁺ cells as previously described [17 (link)]. Briefly, cells were cross-linked in 0.4% formaldehyde in PBS, and lysed (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP40). Lysate was sonicated for 30 cycles of 30 s on/30 s off at 4C° using a Bioruptor Standard sonication device (Diagenode). The cell lysate was then diluted in ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA 150 mM NaCl, 1% Triton, 0.01% SDS) and incubated at 4C° overnight with anti-FOXO3a antibody (Millipore #07–702) and Magna ChIPTM Protein A+G magnetic beads (Millipore #16–663). Beads were then washed (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton, 0.1% SDS) and recovered. The antibody-protein-DNA complexes were reverse cross-linked for DNA isolation and quantitative PCR (qPCR) analysis. Foxo3^-/- TER119⁺ cells were used as negative controls. Putative binding sites were located using MatInspector from Genomatix (http://www.genomatix.de/). Primer specific sequences are listed in S11 Table. See S12 Table for all antibodies.