I’m trying to compare my results to existing ChIP-seq data – I need to continue doing ChIP-seq, right?
Although ChIP-seq and CUT&RUN are distinct protocols, the raw sequencing data are similar, and are processed and visualized using the same tools. Comparing these data sets is straightforward and has been published multiple times in peer-reviewed studies5-8. Furthermore, analysis of ChIP-seq and CUT&RUN data from the same cell line and target show high concordance8. The main difference is that CUT&RUN data have much lower background and require 10-fold fewer cells and sequencing reads.
I already have a good ChIP-seq protocol and/or antibody that works – shouldn’t I stick with it?
Even the best ChIP-seq protocols require more time, cells, and sequencing compared to CUT&RUN. In addition, ChIP-seq is inherently low-throughput, expensive, and has high background – problems that are fully addressed by our CUTANA™ CUT&RUN assay workflow. CUT&RUN also requires minimal optimization for most targets and cell types, in stark contrast to the labor-intensive development of target and tissue-specific cross-linking, fragmentation, and IP conditions for ChIP-seq.
What about my antibody? It works so well in ChIP!
Antibody performance is not a good justification for selecting ChIP-seq. ChIP-grade antibodies are notoriously unreliable, especially for histone PTMs9. EpiCypher found that over 70% of antibodies to histone lysine methylation and acylation PTMs display unacceptable cross-reactivity and/or target efficiency (chromatinantibodies.com). This includes highly cited antibodies for H3K4me3, H3K9me3, H3K27ac, and H3K27me3 – some of the most well-studied marks in chromatin biology. Non-histone PTM targets, such as transcription factors, face similar challenges.
Note: EpiCypher has begun validating antibodies specifically for CUT&RUN applications. Our collection includes antibodies to histone PTMs, transcription factors, chromatin remodeling enzymes, reader proteins, writer enzymes, and more. Check out the list here and subscribe for updates!
You can’t sequence input samples in CUT&RUN (or CUT&Tag). How will I determine enrichment and/or examine nonspecific background?
We recommend running a negative control reaction using an antibody to IgG. IgG is an excellent control for monitoring background and/or nonspecific signal in open chromatin.
To call peaks and determine enrichment, EpiCypher routinely uses MACS210 and SICER11, peak calling programs for ChIP-seq that work well for CUT&RUN12. SICER can be adjusted for analysis of sharp enrichment peaks (e.g. H3K4me3) vs. broad areas of enrichment (e.g. H3K27me3)13. Other options include SEACR14, a peak caller designed for CUT&RUN data, and the CUT&RUNTools 2.0 pipeline, which is designed for CUT&RUN and CUT&Tag data, including analysis of single cells15. It is recommended to test several programs and select the one that faithfully represents the target of interest. See the CUTANA™ CUT&RUN Kit Manual (FAQs section) for more information.