A side-by-side comparison of commercial CUT&RUN kits.

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) followed by next-generation sequencing is a powerful method increasingly used in place of ChIP-seq to profile genome-wide protein-DNA interactions, including those of transcription factors, histone modifications, and chromatin remodelers. Although the choice of antibody is well known to affect data quality, we found that enrichment chemistry and protocol also significantly impact data quality. In this study, we evaluated the performance of three commercially available CUT&RUN kits (Active Motif - ChIC/CUT&RUN Assay Kit, Cell Signaling Technology (CST) - CUT&RUN Assay Kit, EpiCypher - CUTANA™ ChIC/CUT&RUN Kit) on two target histone marks to represent sharp peaks (H3K4me3) and broad domains (H3K27me3). We compared data quality from libraries generated with fresh and frozen cells, as well as with cell numbers at the low and high ends of the recommended input range. Our results show that all three chemistries generate high-quality libraries. Our data revealed that, despite generating fewer H3K4me3 peaks than the EpiCypher and Active Motif workflows, the CST protocol produced libraries with superior signal and higher accuracy. Compared with the H3K4me3 libraries, the H3K27me3 libraries presented a lower percentage of shared peaks across all libraries and lower reproducibility between technical replicates. Compared with the CST protocol, the Active Motif and EpiCypher protocols generated H3K27me3 libraries with higher peak numbers, and the EpiCypher chemistry achieved the highest accuracy. Libraries from cryopreserved cells generated data of similar quality to libraries from fresh cells; libraries generated with 100,000 cells had higher concordance between technical replicates than did libraries made with 500,000 cells.