QuestionsCategory: ChIP-Seq Data AnalysisLibrary ChIP preparation: How do I choose between sonication and enzymatic-based chromatin fragmentation for ChIP-seq? How might this affect sequencing depth?
stonelakeak@nih.gov Staff asked 5 months ago

How do I choose between sonication and enzymatic-based chromatin fragmentation for ChIP-seq? How might this affect sequencing depth?

1 Answers
stonelakeak@nih.gov Staff answered 5 months ago

A more detailed description of sonication and enzymatic-based fragmentation and their benefits are below. Both methods require the same amount of sequencing depth. You should also be aware that when using an enzymatic-based fragmentation method it is preferable to do paired-end sequencing as computational PCR deduplication becomes challenging with this method.
“Sonication, which is the more traditional method used for fragmenting chromatin, uses acoustic energy to forcefully shear the chromatin. Sonicated chromatin works very well for performing ChIP to assess histones and histone modifications, which are abundant and stable components of chromatin. However, over-sonication can damage the chromatin and displace bound transcription factors and cofactors; therefore, sonication typically requires optimization.”
“Enzymatic digestion uses micrococcal nuclease to cut the linker region between nucleosomes. It gently fragments the chromatin and preserves the integrity of chromatin and bound proteins. Therefore, it’s more suitable for performing ChIP to assess transcription factors and cofactors that are less abundant and interact with DNA less stably. In addition, enzymatic digestion provides better reproducibility between experiments compared to sonication. However, over-digestion may lead to loss of nucleosome-free regions.”
Reference : (https://www.cellsignal.com/contents/resources-applications-chromatin-immunoprecipitation/chromatin-ip-frequently-asked-questions/chip-faqs0
– answered by Tovah Markowitz, Paul Schaughency, Vishal Koparde.