First, we investigated whether quantifying DNA directly in freshly prepared, individual chromatin samples would accurately reflect their DNA content and could therefore serve as a basis for determining the amount of solubilized chromatin input for ChIP reactions. Chromatin input was prepared from 30 million fixed K562 cells as previously described [19]. DNA content of the chromatin input (defined as DNAchrom) was directly measured from 0.2% of total input by the Qubit assay [20, 21], a high-sensitivity method specific to double-stranded (ds) DNA, following the manufacturer’s instructions. DNA was then purified from 1% of the total input containing 0.3 to 20 µg of DNAchrom after cross-linking reversal, RNase A and proteinase K treatments, and column purification and quantified by the Qubit assay. The amount of DNA purified from the chromatin input showed strong linear correlation (R2 = 0.99) with DNAchrom (Fig. 1A). Next, we confirmed the linear correlation between the amounts of purified DNA and DNAchrom in 666 different chromatin inputs prepared from various sample types in the context of ChIP experiments (Additional file 1: Table S1). DNAchrom was measured from less than 1% of chromatin input and the measurements typically took less than 5 min to complete. Remarkably, we detected robust linear correlation across all samples and a wide range of purified DNA amounts (0.19 µg to 76.78 µg; R2 = 0.74) (Fig. 1B) despite a variability in the slope of the linear regression obtained in the 6 different sample types analyzed (1.08–2.60; Additional file 2: Fig. S1). Interestingly, we obtained the highest proportional yields of purified DNA in buffy coat and PBMC samples with linear regression slopes of 1.82 and 2.60, respectively (Additional file 2: Fig. S1E and F). These observations may reflect a variability in the reactivity of Qubit reagent with chromatin input or in the efficacy of DNA isolation. Altogether, these results indicate that Qubit assay performed directly in a small fraction of the chromatin input allows quick, easy, and sensitive quantification of the chromatin input immediately after its preparation, enabling the optimization of ChIP antibody:chromatin ratios in individual samples.

Direct measurement of DNA content in chromatin input enables to quantify the amounts of usable input from individual samples. A The DNA content directly measured in chromatin input (DNAchrom) is correlated with the amount of purified DNA. Chromatin input was prepared from fixed K562 cells. DNA amount was directly measured in chromatin input ranging 0.3 – 20 µg by the Qubit dsDNA high sensitivity assay and compared with the amount of purified DNA. The Qubit assay was performed by incubating 2 µl of chromatin input with 198 µl of Qubit reagent. The data was presented as mean ± SD from 2 experiments performed in triplicates. Coefficient of determination (R2) was calculated by the linear regression model. B Correlation between DNAchrom and the amount of chromatin input determined by purified DNA in various sample types. Chromatin input was prepared from cell lines (n = 78), solid tissues (n = 534), and samples derived from peripheral blood (n = 54). DNAchrom was measured from 0.5—1% of chromatin input in individual ChIP reactions and compared with the amount of chromatin input determined by purified DNA

Next, we investigated whether DNAchrom could be used as a reference for determining optimal antibody titers in ChIP applications. A ChIP-seq validated antibody against the histone mark acetylated at histone H3 lysine 27 (H3K27ac) (Abcam, Cat. ab4729), which has been utilized in numerous ChIP-seq experiments to identify active enhancers and promoters [22] (https://www.abcam.com/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html), was selected for the experiment. Chromatin input was prepared from 40 million fixed K562 cells and DNAchrom was measured as described above. 10 μg of DNAchrom was used in individual ChIP reactions with different amounts of antibody ranging from 0.05 to 10.0 µg. The size of the DNA purified from the chromatin input and immunoprecipitated chromatin showed remarkable consistency across the range of antibody titers (Additional file 4: Fig. S3A). ChIP yield, the DNA amount obtained after ChIP divided by the DNA amount of total chromatin input, was measured to access the yield of the immunoprecipitations from individual ChIP reactions. The fold enrichment, the % enrichment of a H3K27ac-positive genomic locus vs. local input (measured by ChIP-qPCR) divided by the enrichment of a H3K27ac-negative locus, was measured to access the specificity of individual reactions. ChIP yield gradually increased from 0.1% to 5.4% (corresponding to about 10 ng to 700 ng of DNA) with increasing amounts of antibody in the reaction (Fig. 2, y-axis on the right). It is noteworthy that 1 ng of ChIP DNA is already sufficient to generate libraries suitable for NGS [19]. In contrast, the fold enrichment of the H3K27ac-positive PABPC1 transcription start site (TSS) locus over the H3K27ac-negative MYT1-TSS locus dramatically decreased from 202- to 18-fold (Fig. 2, y-axis on the left), resulting in an inverse linear correlation between ChIP yield and locus-specific enrichment (R2 = 0.86) (Additional file 4: Fig. S3B). We observed the degree of fold enrichment was dependent on the specific positive and negative genomic loci utilized in the ChIP-qPCR assay as exemplified by the SMARCA4-TSS and other loci (Fig. 2 and Additional file 4: Fig. S3C), but the relationship between fold enrichment and the antibody titer was similar across all loci tested. Based on these observations, we concluded the optimal range of antibody titer was 0.25 μg to 1 μg per 10 μg of DNAchrom, yielding at least 1 ng of purified ChIP DNA and a 5—200-fold enrichment in multiple positive over negative loci. We defined the ratio between the antibody amount and DNAchrom at the optimal titer as “titer 1” (T1, marked as an orange arrow in the figures). It is noteworthy that antibody titer is specific for the antibody lot used in the experiment.

Determination of the optimal antibody titer using DNAchrom. Chromatin input was prepared from 40 million fixed K562 cells and DNAchrom was measured. 0.05 to 10.0 µg of ChIP-validated anti-H3K27ac antibody was incubated with 10 µg of DNAchrom in ChIP reactions. Fold enrichment of H3K27ac-positive PABPC1-TSS and SMARCA4-TSS loci against a negative MYT1-TSS locus was shown in the left y-axis to access the specificity of individual ChIP reaction. The DNA amount obtained after ChIP (ChIP DNA) and ChIP yield were shown in the right Y- axes to access the yield of immunoprecipitation in individual reaction. The optimal titer as the the ratio between antibody and chromatin amounts to yield 1 – 5 ng of ChIP DNA and 5 – 200 fold enrichment in multiple positive over negative loci was highlighted as orange arrow on the top, and the ratio between the antibody amount and DNAchrom at the optimal titer is indicated as titer 1 (T1)

To understand the impact of the antibody titer on the outcome of ChIP experiments and demonstrate the utility of titration-based normalization of antibody amount per available chromatin input, we performed three independent ChIP experiments by incubating various amounts of chromatin input with a fixed amount or normalized amounts of antibody at the optimal titer (Fig. 3A). Chromatin input was prepared from 40 million fixed K562 cells and DNAchrom was measured as described above. Chromatin was diluted to obtain chromatin inputs with various DNAchrom values ranging from 0.3 to 20 μg. Each of these inputs was then incubated with fixed 0.25 μg of antibody (left panel) or the normalized antibody amounts by DNAchrom at the optimal titer of 0.25 μg antibody/10 μg of DNAchrom (T = 1, right panel). Consistent with the results in Fig. 2, ChIP reactions over 10 µg of DNAchrom and the fixed amount of 0.25 µg of antibody (i.e., T = 1 or less) exhibited an enrichment exceeding 100-fold in multiple positive loci with yields below 0.5% (Fig. 3A, left panel). ChIP reactions utilizing less than 10 µg of DNAchrom (i.e., T > 1) showed lower enrichments with higher yields. In contrast, normalized ChIP reactions to the optimal titer (i.e., T = 1) showed similar ChIP yields (