Fig. 4
From: Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments
Titration-based and normalized ChIP-seq approach improves experimental consistency in a large-scale ChIP-seq project. A A schematic diagram of titration-based and normalized ChIP-seq. Chromatin amount is quickly quantitated by the high-sensitivity Qubit assay using less than 0.5% of input. The amount of ChIP-seq validated antibody is normalized to the optimal titer in individual ChIP reaction depending on available amount of DNAchrom. It takes less than 5 min for one antibody where its optimal titer is predetermined, but it may take longer for multiple antibodies B Titration-based and normalized approach generates consistent ChIP yield and peak number over time. Experiments were performed over 1.5-year window as described. Tissue amount (mg), chromatin amount by DNAchrom and purified DNA, ChIP yield, and peak number were plotted with the order of sample processing. The similar y-axis scales with the distribution of chromatin amounts by purified DNA were used for other plots. The solid line in each plot indicates the mean value of measurement. The mean ± SD with the coefficient of variation (CV) was shown in each plot to determine the variability of collected data throughout the samples. C Normalization of antibody titer minimizes the negative correlation of ChIP yield with peak number. Peak number was plotted with % ChIP yield. The solid line indicates the trend line of linear regression. The equation is shown the top. D DNAchrom correlates with DNA amount/mg of tissue. Collected data was sorted based on DNAchrom and plotted with tissue amount (mg), chromatin amount by purified DNA, and DNA amount/mg of tissue. Each dot represents the individual sample from temporal cortex (green) and cerebellum (orange)