Titration-based normalization of antibody amount improves consistency of ChIP-seq experiment

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is most frequently utilized in research areas of chromatin biology and epigenetics. The ENCODE Consortium provided guidelines about antibody but the fundamental aspect regarding antibody titer was not addressed. Here, we introduced a simple and quick DNA-based quantification of chromatin input, allowing to normalize the antibody amount in individual ChIP reaction to the optimal titer. We further demonstrated the titration-based normalization of antibody amount generates improved assay outcome with high consistency among samples in an experiment or recurrent experiments for a broad range of chromatin input amount. Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed for histone H3K27ac mark in the conditions of various titer of antibody in ChIP reactions.. Overall design: Chromatin were prepared from fixed K562 cells. Chromatin amount was aliquoted into 312, 625, 1,250, 2,500, 5,000, 10,000, and 20,000 ng in final 1 ml of ChIP buffer. 0.25 µg of anti-H3K27ac antibody was added to create the ChIP reactions with various antibody titers (the ratios between antibody amount per chromatin amount). Or normalized amounts of the antibody to the optimal titer was added into each reaction.