Fig. 3
From: Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments
Normalized antibody amount to the optimal titer improves the experimental outcome and consistency. A Titration-based normalization of antibody amount to the optimal titer results in consistent ChIP yield and improved specificity. Chromatin input was prepared from fixed K562 cells. Fixed 0.25 µg of anti-H3K27ac antibody (left panel) or normalized amounts to the optimal titer (right panel) were incubated with DNAchrom ranging 0.31 – 20 µg in ChIP reactions. The ratio of antibody amount/DNAchrom at the optimal titer is indicated as 1 (orange arrow), and the relative antibody titers ranging 0.5 to 32 is shown on the top. The fold enrichments of positive loci over a negative locus (left Y-axis) were plotted with the amounts of chromatin input by DNAchrom. The % ChIP yield (right Y-axis) was calculated by DNAs isolated from chromatin input and ChIPed chromatin. The optimal titer determined in Fig. 2 was highlighted as orange arrow on the top. The experiments were performed with triplicates. B-C Normalization of antibody amount to the optimal titer improves peak number and consistency. The libraries were generated from the experiments described above. The peaks were called by Macs2 using FDR < 0.01. (B). The peak numbers obtained with fixed or normalized antibody amounts were plotted with the amounts of chromatin input by DNAchrom. (C). The peak numbers were plotted for antibody titers in the reactions with the fixed amount of antibody. The dotted line indicates the peak numbers after the normalization of antibody amount to the optimal titer when the same amounts of DNAchrom are available. D Heap map analysis of peaks around transcription start sites. Read intensities of the peaks around ± 5 Kb of TSSs were visualized by Partek Flow software. The library ID indicates DNAchrom (µg)-antibody titer in ChIP reactions. The reactions that the optimal titer was determined in Fig. 2 were marked with orange. E The representative snapshot image of ChIP-seq results generated with various antibody titers and after normalizing antibody amount to the optimal titer. The read densities were visualized in a 212 kb genomic region around the ACTB gene using the Integrative Genomics Viewer