NPAS2 ChIP-on-chip in MCF7 cells

The aim of the current study is to create a transcriptional profile of genes regulated by the circadian gene NPAS2, using a human binding ChIP-on-chip analysis of NPAS2 in MCF-7 cells. The ChIP assay was performed using the ChIP Assay Kit ((Millipore, Billerica, MA) according to the manufacturer’s protocol. Briefly, cells were grown in 100 mm cell culture plates to 80% confluence. Cross-linking of protein and DNA was performed using cell culture medium containing 1% formaldehyde at room temperature for 10 minutes. Sonication was performed using an Omni Ruptor 250 Ultrasonic Homogenizer (omni International, Marietta, GA) to generate input material. This input sample was then incubated with primary antibody (anti-NPAS2) and negative control (Rabbit IgG) for immunoprecipitation (IP). Agarose beads were added to the reactions to bind protein-conjugated antibody. The antibody-protein-DNA complex was then eluted from the agarose beads with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) and the cross-linking was reversed by adding 5M NaCl for 4 hours at 65ºC followed by proteinase K digestion at 45ºC for one hour. The final DNA, representing NPAS2 target sequences, was purified using the GENECLEAN Turbo kit (Qbiogene, Carlsbad, CA) according to the manufacturer’s instructions. Ligation-mediated PCR (LM-PCR) (Agilent Technologies, Foster City, CA) was used to amplify DNA according to the manufacturer’s protocol. Two ChIP assays were performed independently and both amplified DNA samples were used in the subsequent human binding microarray analysis. Binding microarray analysis We worked with MOgene Inc (St Louis, MO) to perform the Human Binding Array (Agilent) using the amplified DNA obtained from the ChIP experiment. Labeling, hybridization, and image analyses were all performed at MOgene, an Agilent certified service provider for ChIP-on-chip analysis. The binding array includes a total of 488k probes that target ~17,000 of the best defined human transcripts covering 5.5 kb upstream to 2.5 kb downstream from the transcriptional start sites. A whole-chip error model was used for array data analysis and to call a bound gene (target gene). The neighborhood error model takes into account neighboring probes and calls a region as bound, not just a single probe. Genes associated with significant regions are therefore considered target genes. The binding array analysis was performed for both duplicate samples from the ChIP assay, and since the array requires two slides per sample, there are four total samples included in this submission