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:

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

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