To mock or not: A comprehensive comparison of mock IP and DNA input for ChIP-seq

Jinrui Xu, Michelle M. Kudron, Alec Victorsen, Jiahao Gao, Haneen N. Ammouri, Fabio C.P. Navarro, Louis Gevirtzman, Robert H. Waterston, Kevin P. White, Valerie Reinke, Mark Gerstein

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect transcription-factor (TF) binding sites in the genome. Its success depends on appropriate controls removing systematic biases. The predominantly used controls, i.e. DNA input, correct for uneven sonication, but not for nonspecific interactions of the IP antibody. Another type of controls, 'mock' IP, corrects for both of the issues, but is not widely used because it is considered susceptible to technical noise. The tradeoff between the two control types has not been investigated systematically. Therefore, we generated comparable DNA input and mock IP experiments. Because mock IPs contain only nonspecific interactions, the sites predicted from them using DNA input indicate the spurious-site abundance. This abundance is highly correlated with the 'genomic activity' (e.g. chromatin openness). In particular, compared to cell lines, complex samples such as whole organisms have more spurious sites-probably because they contain multiple cell types, resulting in more expressed genes and more open chromatin. Consequently, DNA input and mock IP controls performed similarly for cell lines, whereas for complex samples, mock IP substantially reduced the number of spurious sites. However, DNA input is still informative; thus, we developed a simple framework integrating both controls, improving binding site detection.

Original languageEnglish (US)
Article numbere17
JournalNucleic acids research
Issue number3
StatePublished - Feb 22 2021
Externally publishedYes

Bibliographical note

Funding Information:
National Institutes ofHealth grant [U41HG007355]. Funding for open access charge: U41HG007355.

Publisher Copyright:
© 2021 The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.


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