ChIP assays were performed using SKNO-1 cells, the antibody against CBFb and optimized primer pairs for qPCR. Sheared chromatin from 1.25 million cells and 4 ul of antibody were used per ChIP experiment. QPCR was performed using primers specific for the FUT7, OGG1, NFE2, and SPI1 genes. Image shows the relative occupancy, calculated as the ratio + control/background for which the MYOG gene was used.
Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against human CBFb. The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:8, 800.
ChIP was performed as described above. The IP'd DNA from 6 ChIP's was pooled and analysed with an Illumina Genome Analyzer. The 32 bp tags were aligned to the human reference genome (hg18) using the ELAND algorithm. Image shows the results of the complete chromosome 3 and three genomic regions region surrounding the OGG1, FUT7 and NFE2 genes, respectively. The position of the PCR amplicon is indicated with an arrow.