If your CUT&RUN sequencing libraries contain large fragments (>500 bp) on Bioanalyzer/TapeStation traces, it is likely due to one of the following reasons:
Poor sample quality
If starting cells have poor morphology, low viability, and/or are lysing, you may observe higher background and potential enrichment of large fragments. This is also true for samples that have been cross-linked; cells should remain intact after cross-linking. Review experimental success metrics and reference sample prep protocols and variations as applicable.
If cell lysis is a problem, or if cells appear sensitive to CUT&RUN Wash Buffers, consider using nuclei for CUT&RUN experiments.
Inefficient ConA bead binding
If cells are not bound to ConA beads with >95% efficiency, you may observe higher background and potential enrichment of large fragments. Always check that the majority of cells (or nuclei) are bound to ConA beads.
Increased rates of cell lysis can also reduce ConA bead binding efficiency and cause clumping of beads, leading to increased background and large fragments. As above, working with nuclei may alleviate this issue.
Incomplete cell permeabilization
Permeabilization is critical when using whole cells for CUT&RUN. If cells are not efficiently permeabilized, antibodies and pAG-MNase will be poorly distributed in your CUT&RUN reactions, resulting in larger fragments. Optimize Digitonin permeabilization conditions for each cell type before starting CUT&RUN.
Alternatively, you can use extracted nuclei, which do not require Digitonin optimization (use 0.01% Digitonin in buffers).
Library prep PCR steps were incorrect
Issues with large fragment enrichment often indicate problems with CUT&RUN Library Prep PCR. Be sure to follow the PCR parameters in our protocols and manuals. Our library prep PCR conditions are optimized for 200-700 bp fragments and should eliminate large fragments.