Enrichment of large peaks (>700 bp) in CUT&Tag sequencing libraries can be caused by multiple factors, outlined in detail below. We recommend repeating the experiment and paying close attention to sample prep steps, bead resuspension during tagmentation and PCR, and confirming PCR cycling parameters match those in the CUTANA™ CUT&Tag Kit Manual and DIY Protocol.
Poor sample prep and/or poor nuclei extraction. Poor sample prep can result in issues with ConA bead binding, bead clumping, and sample loss, affecting the entire CUT&Tag workflow. Review quality control checks for starting cells, the nuclei extraction protocol, and reference sample prep steps and variations if applicable.
Problems with ConA bead resuspension. Excessive bead clumping impacts CUT&Tag data quality. It is especially critical to keep ConA beads resuspended during pAG-Tn5 binding, tagmentation, and indexing PCR. Proper bead mixing helps ensure even distribution of pAG-Tn5 across nuclei, robust Tn5 activation, and maximizes PCR efficiency.
Incorrect PCR cycling parameters and/or excessive amplification. Be sure to strictly follow the PCR steps as described in our CUT&Tag Kit Manual and DIY Protocol. The PCR parameters outlined are optimized for 200-700 bp fragments and should eliminate large fragments. If the PCR parameters are correct, but you are still enrichinig large fragments, repeat the CUT&Tag assay using fewer PCR cycles.