Illumina recommends 5–10 ng of ChIP-enriched, fragmented input DNA.
Input DNA Quantitation
Correct quantification of ChIP DNA is essential. The ultimate success or failure of a library preparation strongly depends on using an accurately quantified amount of input DNA.
It is difficult to accurately measure the ChIP DNA starting amount, because the yield is low (<10 ng).
Methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Reference literature or other sources for recommendations.
Illumina recommends using fluorometric-based methods for quantification including Qubit or PicoGreen to provide accurate quantification for ChIP DNA. UV-spec based methods, such as the Nanodrop, will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides, which can give an inaccurate measurement of ChIP DNA.
DNA quantification methods that rely on intercalating fluorescent dyes measure only dsDNA and are less subject to excess nucleic acids. However, these methods require the preparation of calibration curves and are highly sensitive to pipetting error. Ensure that pipettes are correctly calibrated and are not used at the volume extremes of their performance specifications.
Assessing DNA Quality
Absorbance measurements at 260 nm are commonly used to assess DNA quality. Use the following guidelines:
The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity; values of 1.8–2.0 are indicate relatively pure DNA.
The presence of RNA or small nucleic acid fragments, such as nucleotides, can compromise absorbance measurements.
Carefully collect ChIP DNA samples to make sure that they are free of contaminants.
A further validation step can be performed using the Agilent Bioanalyzer with a High Sensitivity Chip for the correct ChIP DNA size distribution, presence of contaminants, etc.