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.
Troubleshooting low CUT&RUN DNA yields
If your experiment failed or if you have very low yields (< 5 ng), see this article and consider the following questions:
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 sample prep and ConA bead binding?
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.
Confirm proper ConA bead storage conditions and quality. Are ConA beads brown and stored at 4˚C? ConA beads should NEVER be frozen.
Have reactions been mixed properly using a nutator? Have ConA beads become clumpy or dried out during the protocol?
Figure 1. Overview of CUTANA CUT&RUN quality control metrics.
Troubleshooting sequencing library yields and fragment distribution
Concern | Potential causes & troubleshooting approach |
|---|---|
Low library yields, no enrichment in Bioanalyzer or TapeStation results | ⚠ Low CUT&RUN yields, low inputs for library prep
⚠ Library prep technique
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Adapter dimers | ⚠ Self-ligation of sequencing adapters, preferentially amplified due to their small size
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Troubleshooting CUT&RUN sequencing and results
Concern | Cause & troubleshooting approaches |
|---|---|
Sequencing a low-concentration DNA library | ⚠ If it is not possible to repeat library prep:
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Background in open chromatin | ⚠ Indicates over-digestion by MNase
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Experimental target shows high background and/or is indistinguishable from IgG negative control | ⚠ Over-digestion by MNase, DNA damage, antibody failure. Use 500,000 cells per reaction; include reactions with control antibodies and the K-MetStat Panel. General tips:
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