What are considerations for library prep and sequencing from low yields?
Ideally, you would troubleshoot low CUT&RUN yields, repeat the experiment, and then perform library prep and sequence. This will result in higher quality sequencing data compared to using ultra-low CUT&RUN yields. However, in some cases, repeating the experiment isn’t possible. In fact, for some targets and cell types, low CUT&RUN yields are unavoidable.
Although useful sequencing data can be obtained, the resulting libraries often have low concentrations with elevated adapter dimers, reduced read diversity, and low signal over background, all of which impact data quality.
Use the CUTANA CUT&RUN Library Prep Kit (EpiCypher 14-1001 & 14-1002), which is specifically optimized for CUT&RUN workflows and includes guidelines for library prep from low CUT&RUN yields (see manual at epicypher.com/protocols).
- Remove adapter dimers that have greater than 5% “%Integrated area” (as called by TapeStation analysis), meaning that the adapter dimers comprise >5% of your library. High levels of adapter dimers take up valuable sequencing bandwidth. See our articles on avoiding or removing adapter dimers for more.
- Increase number of cycles for indexing PCR to improve library yields for Bioanalyzer/TapeStation analysis and sequencing. Refer to the CUTANA CUT&RUN Library Prep Kit manual for guidance.
- Deeper sequencing (>8 million reads) is recommended to capture read diversity.
- Read duplicates may be increased by some of these strategies, but can be filtered out with Picard (broadinstitute.github.io/picard).