How to design and optimize CUT&RUN assays
Like all experiments, CUT&RUN requires thoughtful experimental design.
Some steps may require user-specific optimization. For ease, we have broken these down into four main steps:
I. Sample Preparation
Number of Cells. We recommend starting with 500,000 native (unfixed) cells, particularly when mapping new targets or using new cell types.
- Notes on cell types: EpiCypher has developed several protocol variations to ensure reliable CUT&RUN data from diverse biological samples. For recommendations on alternate sample prep protocols (e.g. using adherent cells, tissues, immune cells, nuclei, and frozen cells/nuclei), see this page.
- Notes on using tissues: CUTANA CUT&RUN Kits and protocols are compatible with tissues. The primary requirement when using tissues is to process samples into a monodispersion of cells or nuclei. See this article for more information.
- Notes on low inputs: Following initial validation of workflows using 500,000 cells and control antibodies, cell numbers can be reduced following the recommendations described here.
Replicates. CUT&RUN is very robust and reliable – two biological replicates (e.g. same cell type harvested from two mutant mice) per target are usually sufficient.
Cross-linking. CUT&RUN is a native technique, meaning that it performs best on unfixed cells (or nuclei). This is a major advantage compared to ChIP, which typically requires heavy cross-linking to stabilize target associations with DNA. Cross-linking and chromatin fragmentation are major contributors to high background, low yields, artifacts, and/or data variability in ChIP. These steps aren’t required for CUT&RUN, which streamlines the assay, maximizes on-target DNA recovery, and allows for reduced cell numbers.
However, there are some instances where light cross-linking can be useful in CUT&RUN, by localizing and stabilizing potentially labile PTMs (e.g. histone acetylation) and acetyl-binding proteins (e.g. bromodomains) or when performing time-course or drug treatment assays. Note that we always recommend trying native conditions first, or at least in parallel with cross-linked samples. See this blog and our cross-linking protocol for detailed information.
Optimization of Cell Permeabilization. This is a key step of the CUT&RUN protocol, as the cell membrane must be pervious to antibodies and pAG-MNase, yet intact enough to prevent cell lysis. EpiCypher's standard CUT&RUN condition for whole cells is 0.01% Digitonin. This may not be sufficient dependeing on cell type (e.g. fibroblast or macrophages). For optimizing Digitonin conditions, use the full step-by-step procedure as outlined here. If you are still experiencing permeabilization issues, try extracting nuclei from your cells.
II. Assay Controls
All CUT&RUN experiments should include appropriate controls, provided in the CUTANA™ CUT&RUN Kit, to evaluate assay success and individual reaction performance.
Quality Control Checks. EpiCypher has incorporated multiple quality control check points to help ensure assay success. For instance, we have outlined a simple Trypan Blue staining protocol to confirm sample binding to ConA beads prior to antibody addition – a critical step of CUT&RUN. A full list of quality control checks, both before AND after sequencing, can be found here.
Spike-in controls. Spike-in controls are essential for all genomics assays. The CUTANA CUT&RUN Kit includes includes E. coli spike-in DNA, which can be added to all reactions as a control for library prep and to aid in sequencing normalization.
For reactions targeting histone PTMs, EpiCypher offers SNAP-CUTANA™ Spike-in Controls. SNAP-CUTANA Spike-in Controls are panels of highly pure nucleosomes, each containing a defined histone PTM and accompanying PTM-specific DNA barcode. The nucleosomes come pre-bound to magnetic beads for simple one-step addition to CUT&RUN workflows, allowing users to examine antibody specificity, signal over background, and assay variability. Panels are currently available for histone lysine methylation PTMs (K-MetStat Panel) and lysine acetylation and extended acyl states are coming soon. Check to make sure that your target is included in the panel before adding to reactions; see our section on SNAP-CUTANA Spike-ins to learn more about how to leverage SNAP-CUTANA Spike-ins for your workflow.
Positive and Negative Control Reactions. Reactions using negative control (IgG) and positive control (H3K4me3) antibodies should be included in every experiment to validate CUT&RUN workflows. EpiCypher also recommends adding the SNAP-CUTANA™ K-MetStat Panel of spike-in controls to these positive and negative control reactions, to provide a direct readout of assay success and to guide troubleshooting experiments. For more information about the SNAP-CUTANA K-MetStat Panel and how it can be used to guide troubleshooting, see this article.
III. Antibody Selection
Select a target-specific antibody. As with ChIP-seq, a quality antibody is essential for generating robust and reliable genomic profiles. Key considerations include:
- Performance in other assays, such as ChIP, does NOT guarantee success in CUT&RUN! The antibody should demonstrate high-specificity (low cross-reactivity) and high efficiency (comprehensive target recognition) in CUT&RUN.
- EpiCypher offers CUTANA™ CUT&RUN antibodies against histone PTMs and multiple target classes of chromatin-associated proteins. These antibodies have been extensively screened for robust performance in CUT&RUN assays.
- When analyzing a new target in the absence of validated antibodies, we recommend sourcing 3-5 antibodies from various reputable vendors that bind unique epitopes. Test these antibodies in parallel CUT&RUN assays with cells known to express the target protein. Then, select the best antibody based on overall yield, signal over background, and peak structure/enrichment.
- Need more information? We cover principles for antibody selection extensively here.
IV. DNA Purification and Analysis
DNA purification. Yields from CUT&RUN are much lower compared to ChIP, and DNA fragments can also be much smaller, particularly for transcription factors. In these cases, it is important to use a DNA purification protocol that is optimized for low DNA concentrations and small fragment sizes. EpiCypher’s CUTANA CUT&RUN Kit includes DNA purification protocols that are specifically designed to capture small fragments and maximize DNA concentration for subsequent library prep.
- Note: We also offer these DNA purification reagents in a separate CUTANA™ DNA Purification Kit!
Library prep. Library prep is a standard part of genomics assays and there are numerous kits and multiplexing strategies available. However, the low yields and small fragments from CUT&RUN make it difficult to utilize existing library prep kits. For instance, it isn’t clear if adapter concentrations should be adjusted for low DNA inputs, or the optimal SPRI bead ratio to use for enriching small CUT&RUN fragments while avoiding adapter-dimer contaminants. To address these concerns, EpiCypher has launched an all-inclusive Library Prep Kit specifically optimized for CUTANA CUT&RUN assays. You can read more about library kit development in this blog post.
Sequencing depth. The number of sequencing reads depends on several factors, including the number of cells, target abundance, and antibody quality. For most targets, 3-8 million paired-end reads are sufficient and will allow you to multiplex more samples per run. If you have further questions about sequencing depths or CUT&RUN library prep, see this article.