Troubleshooting overview
There are many steps to consider when troubleshooting CUT&RUN assays. A good place to start is by reviewing the basic CUT&RUN troubleshooting guidelines below. Alternatively, navigate to the specific topics.
Basic CUT&RUN troubleshooting guidelines:
See the quality control metrics in the Figure below. If your experiment failed or if you have very low yields (< 5ng), see this article and consider the following questions:
- What is your cell/sample type? Review sample prep protocol modifications.
- 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 & the K-MetStat Panel? These controls are crucial for troubleshooting CUT&RUN.
- Confirm proper ConA bead storage and condition. 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. Overview of CUTANA™ CUT&RUN kit quality ensure metrics and checks to ensure successful chromatin profiling. Quality control metrics are listed for each section of the CUT&RUN workflow.
Troubleshooting fragment distribution and library yields (after library prep)
| Concern | Potential causes & troubleshooting approach |
|---|---|
| Low library yields, no enrichment in Bioanalyzer or TapeStation results | ⚠ Low CUT&RUN yields, low inputs for library prep • See this article for guidance ⚠ Library prep technique • Use the EpiCypher CUTANA CUT&RUN Library Prep Kit (EpiCypher 14-1001 & 14-1002), specifically developed for CUT&RUN |
| Adapter dimers | ⚠ Self-ligation of sequencing adapters, preferentially amplified due to their small size (see this FAQ) • Keep adapter ligation reagents on ice during ligation setup • Remove adapter dimers comprising >5% of a library; see the CUT&RUN Library Prep Kit Manual |
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: • Use a Speedvac to increase the library concentration • Add as much of the library as possible to the sequencing pool • Deeper sequencing is recommended |
| Background in open chromatin | ⚠ Indicates over-digestion by MNase • Repeat assay with fresh buffers • Make sure MNase digestion is performed on ice |
| 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 & the K-MetStat Panel. General tips: • Process cells quickly and resuspend in cold Antibody Buffer • Test multiple antibodies to experimental target • Ensure MNase digestion is incubated on ice for 2 hours • Keep adapter ligation reagents on ice during ligation setup |
