8 basic steps of CUT&RUN
CUT&RUN consists of just a few basic steps!
Step 1: Isolate cells & immobilize to concanavalin A (ConA) beads
Cells (or nuclei) are bound to magnetic beads coated with Concanavalin A (ConA), a lectin that binds to cell surface proteins. This supports high-throughput formatting and simplifies the separation of cells from clipped chromatin fragments in later steps.
It is important to avoid bead dry out and clumping, as this results in sample loss and reduces yields. Important quality control checks to confirm cell integrity and ConA bead binding can be found here. Additionally, some sample types may require modifications; see Sample Prep for more information.
Step 2: Permeabilize cells
Immobilized cells are treated with a buffer containing Digitonin, a nonionic detergent that permeabilizes cell membranes at low concentrations. Permeabilization is crucial for antibody and pAG-MNase binding, and allows MNase-digested DNA to diffuse into solution in later steps. Digitonin conditions must be optimized for each cell type to avoid cell lysis and/or incomplete permeabilization. For information about optimization of this step click here.
Step 3: Incubation with target-specific antibody
An antibody to the target of interest is added to the reaction and incubated overnight at 4˚C. We suggest including negative control (e.g. IgG) and positive control (e.g. H3K4me3) reactions in every experiment.
The specificity and binding efficiency of antibodies is crucial for successful CUT&RUN. In fact, the background of this assay is so low that an antibody with poor efficiency will not generate high enough yields for PCR and sequencing. In contrast, a nonspecific antibody may provide decent yields, but will lead to incorrect biological interpretations. See additional notes on antibody selection and assay controls to learn more.
Step 4: pAG-MNase binding
The following day, bead-bound cells are washed to remove unbound and/or non-specifically bound antibody. pAG-MNase is added to the reaction in the absence of calcium (Ca2+) to prevent premature activation of MNase. The immunoglobulin binding properities of pAG act to "tether" MNase to antibody-bound chromatin. Following pAG-MNase incubation, the cell/bead mixture is washed several times to remove excess pAG-MNase, which helps to prevent non-specific cleavage.
Step 5: pAG-MNase activation
Ca2+ is added to the reaction to activate MNase, which cleaves DNA proximal to where the antibody is bound. Cleaved chromatin fragments diffuse into the supernatant, while remaining bulk chromatin remains inside the bead-immobilized cells.
Because MNase is a processive enzyme, the reaction must be quenched to prevent over-digestion of released DNA. Following pAG-MNase incubation, a Stop Buffer containing EDTA and EGTA is added to chelate free calcium ions and halt enzymatic activity. Reactions are briefly heated to degrade RNA and release any remaining chromatin fragments into solution.
Step 6: DNA purification
Isolation of CUT&RUN enriched DNA is straightforward because the cells remain bound to magnetic ConA beads. Bead-coupled cells containing bulk chromatin are magnetically separated from the clipped target DNA, which remains in solution. Target DNA is purified and quantified with a fluorometric assay (e.g. ThermoFisher Qubit™).
DNA yields should NOT be used as an indicator of CUT&RUN success. Instead aim for ~5 ng DNA, which will allow robust CUT&RUN library prep. It is NOT recommended to analyze raw CUT&RUN DNA on the Bioanalyzer/TapeStation, as yields are often below the limit of detection for these methods.
EpiCypher also checks that the yields from positive controls and experimental targets are greater than the IgG negative control reaction, even if only slightly higher. After confirming DNA quality according to our outlined metrics, proceed to library prep.
Step 7: CUT&RUN library prep
Purified CUT&RUN DNA is repaired, ligated to sequencing adapters, and PCR-amplified to generate sequencing libraries. PCR is performed using parameters optimized for low CUT&RUN yields and small fragment sizes, and barcoded primers are used to enable multiplexed sequencing. EpiCypher’s Library Prep Kit is specifically optimized to further streamline your workflow.
Prior to sequencing, the best method to confirm CUT&RUN success is fragment size distribution analysis of purified libraries. The fragment size distribution and concentration of CUT&RUN libraries is confirmed using capillary electrophoresis (e.g. Agilent Bioanalyzer or TapeStation). Because MNase digests fragments to nucleosome-level resolution, the average peak size is typically ~300 bp (~170 bp fragmented DNA + adapters). For more further reading on assuring sequencing library quality, see this article.
Step 8: Illumina® next-generation sequencing
Libraries are pooled at equimolar ratios and loaded onto the desired platform for sequencing. Only 3-8 million reads per sample are required for robust signal over background (vs. >20 million for ChIP-seq), allowing users to multiplex 10s-100s of samples in a single run.
