CUT&Tag consists of just a few basic steps!
Step 1: Isolate nuclei and immobilize to ConA beads
Nuclei are prepared from bulk cell populations and coupled to magnetic beads coated with Concanavalin A (ConA), a lectin that binds cell surface proteins. Immobilization of nuclei on beads allows the use of 8-strip tubes and multi-channel pipettors, streamlining the workflow and increasing throughput.
High quality sample prep is essential to CUT&Tag success. For instance, lysed nuclei can cause ConA bead clumping, making it difficult to mix reactions and reducing assay yields. To help ensure robust sample prep, we have developed a series of quality control checks to (1) determine the viability and morphology of starting cells, (2) examine the integrity of isolated nuclei, and (3) confirm nuclei binding to ConA beads. These checks, along with modifications for unique sample types, are outlined in the Sample Prep Protocols and Related FAQs section.
Important note: Avoid ConA bead dry out and clumping, particularly during pAG-Tn5 binding and tagmentation, as this results in sample loss and reduces yields. High quality sample prep can help reduce clumping; see the CUTANA CUT&Tag Kit manual (or DIY CUT&Tag protocol) for step-by-step guidance on ConA bead resuspension.
Step 2: Incubate with target-specific primary antibody.
An antibody specific to the histone PTM of interest is added to the reaction and incubated overnight at 4˚C. Negative control (e.g. IgG) and positive control (e.g. H3K4me3 and H3K27me3) reactions should be included in every CUT&Tag experiment.
Selection of a highly specific antibody is critical for CUT&Tag assay success, as off-target binding can significantly impact data quality and biological interpretations. The low background of CUT&Tag also makes antibody binding efficiency important. For instance, a low-efficiency antibody may struggle to generate high enough CUT&Tag yields for next-generation sequencing. See the Antibody Validation and FAQs section to learn more.
Step 3: Incubation with a species-specific secondary antibody
The following day, reactions are washed and labelled with a species-matched secondary antibody (e.g. anti-rabbit, anti-mouse). This step increases the number of pAG-Tn5 binding sites, amplifying Tn5 localization and on-target signal in sequencing data. At the end of this step, reactions are washed with a high-salt buffer to remove unbound antibodies and prepare for tagmentation. The high-salt wash is essential to minimize nonspecific binding of pAG-Tn5 to accessible DNA, which results in ATAC-like signal in CUT&Tag datasets.
Important note: The cell-bead mixture often becomes clumpy after overnight incubation at 4˚C. See the CUTANA CUT&Tag Kit manual (or DIY CUT&Tag protocol) for guidance at these steps.
Step 4: Incubate with pAG-Tn5
pAG-Tn5, pre-loaded with sequencing adapters, is added to each reaction and binds antibody-labeled chromatin via the immunoglobulin binding properties of pAG. As noted above, it is important that ConA beads are resuspended during pAG-Tn5 incubation to maximize binding. Following incubation, the reactions are washed several times using a high-salt buffer to remove nonspecific pAG-Tn5.
Step 5: pAG-Tn5 activation and targeted tagmentation
Tn5 is activated by addition of magnesium (Tagmentation Buffer) to cleave and ligate sequencing adapters to DNA proximal to antibody-bound chromatin. These steps are performed under high salt to minimize nonspecific Tn5 cleavage and/or ATAC-like signal. Rinsing the beads with Pre-Wash Buffer effectively quenches the tagmentation reaction.
SDS Release Buffer is added to digest proteins and release tagmented DNA into solution. Finally, SDS Quench Buffer is added to neutralize SDS and enable downstream indexing PCR.
Important note: SDS inhibits indexing PCR, so be sure to quench SDS activity!
Step 6: Indexing PCR and library cleanup
Indexing PCR is performed to add indexes (i.e. barcodes) to tagmented DNA, resulting in uniquely barcoded CUT&Tag libraries for multiplexed sequencing. Notably, in EpiCypher’s exclusive Direct-to-PCR CUT&Tag protocol, sequencing libraries are generated using the entire reaction mixture from Step 5; ConA beads are not removed and there is no DNA purification step prior to PCR.
During PCR, indexing primers anneal to ligated adapter sequences, ensuring selective amplification of tagmented DNA — even in the presence of cell debris. PCR parameters are specifically optimized for mononucleosome-sized fragments, and primers contain barcodes (or indexes) to enable multiplexed sequencing. This strategy allows you to go from cells to PCR-amplified sequencing libraries in a single tube, minimizing sample loss and streamlining library prep.
Libraries are purified using a SPRI-bead approach, optimized for 8-strip tubes.
Important note: The number of PCR cycles may require optimization; see CUT&Tag Optimization and Troubleshooting for guidance.
Step 7: Analysis of library quality
CUT&Tag sequencing libraries are purified using SPRI magnetic beads. Library concentration is determined using a fluorometric assay (e.g. ThermoFisher Qubit™) and fragment size distribution is examined by capillary electrophoresis (e.g. Agilent Bioanalyzer® or TapeStation®). The PCR parameters in this protocol amplify fragments compatible with Illumina® paired-end sequencing, with an average fragment size of ~300 bp (~170 bp nucleosome + sequencing adapters). When comparing library yields and distribution from CUTANA™ CUT&Tag assays, keep the following points in mind:
The BEST indicator of CUT&Tag success prior to sequencing is predominant enrichment of mononucleosome-sized fragments (~300 bp) in Bioanalyzer or TapeStation traces.
Library yields should NOT be used as a definitive metric of success because results vary by cell type, target abundance, antibody specificity, number of nuclei, and so forth. Instead, EpiCypher recommends aiming for ~30 ng total DNA, which allows accurate library quantification, minimizes PCR duplicates, and enables library pooling at standard concentrations.
Step 8: Next-generation sequencing
Libraries are pooled at equimolar ratios and loaded onto the desired Illumina platform for sequencing. Only 5-8 million total sequencing reads per reaction are required for robust signal over background, compared with >20 million for ChIP-seq, allowing users to multiplex more libraries per run. The two versions of our CUTANA CUT&Tag Kit (EpiCypher 14-1102 and 14-1103) contain distinct indexing primer sets that, combined, generate up to 96 uniquely barcoded sequencing libraries.