title: Illumina HiSeq 2000 sequencing; ZMYM2-WT, ZMYM2-SIM2mut, and FOXO3 ChIP-seq in a U2OS stable cell line
design: ZMYM2-WT, ZMYM2-SIM2mut, and FOXO3 ChIP-seq in a U2OS stable cell line
library_construction_protocol: After reverse cross-link and protein digestion with Proteinase K (Roche), DNA was purified using Qiaquick, PCR purification kit (Qiagen) following the manufacturer's instructions. The stable cell lines were grown in DMEM supplement with 10% foetal bovine serum. Media contained 100 micro-g/ml of Hygromycin. Expression of ZMYM2(WT) or ZMYM2(SIM2mut) was induced by treating the cells for 48 hours with 100 ng/ml [for ZMYM2(WT)] or 50 ng/ml [for ZMYM2(SIM2mut)] doxycycline (Sigma). Expression of FOXO3 was induced by treating the cells for 48 hours with 1 ng/ml doxycycline (Sigma). Cells were double cross-linked with dimethyl adipimidate (DMA) for 20 minutes and subsequently with Formaldehyde for 10 minutes. ChIP-seq was performed as described in Schmidt et al., 2009, using 60 x 106 U2OS-ZMYM2(WT) or U2OS-ZMYM2(SIM2mut). Immuno-precipitation was performed overnight at 4 degrees C by incubating the shared DNA-protein complex with 6 ug of anti-Flag (Sigma) or control IgG (Millipore) antibodies previously coupled to Dynabeads protein G (Invitrogen). Schmidt D, Wilson MD, Spyrou C, Brown GD, Hadfield J, Odom DT. (2009)  ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 48: 240-248. Note: paired-end sequencing libraires were created in this experiment but only the forward (R1) reads are available in this submission.  The 'LIBRARY_LAYOUT' attribute is therefore set to be 'SINGLE' rather than 'PAIRED'.
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: U2OS-Flp-in-FOXO3