Below we outline the main areas of concern and general considerations for troubleshooting CUTANA™ CUT&RUN assays. For ALL troubleshooting, we recommend reviewing the basic quality control metrics in Figure 1.
What is your cell/sample type? See Sample Prep section for specific recommendations.
Are Digitonin permeabilization conditions optimized for your cell type?
Have you confirmed ConA bead binding?
Are ConA beads brown and stored at 4˚C? ConA beads should NEVER be frozen.
Are you using the recommended 500,000 cells per reaction? Success from low cell numbers depends on antibody quality and target abundance. Review this article for guidance.
Have you included reactions with control antibodies and the K-MetStat Panel? These controls are crucial for troubleshooting CUT&RUN, as outlined here.
Have reactions been mixed properly using a nutator? End-over-end rotation can result in ConA beads becoming clumpy or dried out during the protocol, which is why traditional rotators should be avoided.
Did you notice significant clumping throughout the CUT&RUN protocol? If so, we recommend using the CUTANA™ Wash Buffer Enhancer Set (EpiCypher 14-1803) in your starting buffers.
If your continue to observe very low yields (< 5 ng) despite following the above guidelines, see this article.
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Figure 1. Overview of CUTANA CUT&RUN quality control metrics.