CUTANA™ CUT&RUN Services

CUTANA CUT&RUN chromatin mapping services use automated, quality-controlled workflows to enable medium to large-scale analysis of transcription factors, histone PTMs, and other chromatin-associated targets. Combined with the improved costs and sensitivity of CUT&RUN (compared to ChIP-seq), epigenomic mapping can now be applied at a greater scale than ever before. From early-stage pilot studies requiring optimization for new sample types and targets, to large-scale multi-center collaborations, or drug screens profiling thousands of samples, our genomics experts can help find solutions.

If you are interested, fill out the form above. Our experts can help you estimate total project size and determine if CUT&RUN Services is right for you. If you have a smaller project and/or the capability to bring CUT&RUN in-house, we offer user-friendly CUTANA CUT&RUN and Library Prep Kits, which include all reagents and controls needed for 48 CUT&RUN reactions.

Please note: CUT&RUN Services are for Research Use Only (RUO) as our laboratory is not CLIA certified.

Our CUT&RUN Service protocols are optimized for freshly isolated and frozen cells or nuclei from various sources. For certain targets, such as labile histone lysine acetylation PTMs or acetyl-binding proteins, light cross-linking may be recommended. EpiCypher has successfully used our automated CUT&RUN assays for a variety of sample types and cell preparations, including:

• Suspension and adherent cell lines
• Drug-treated or stimulated cells
• Primary cells, including immune cells
• FACS-isolated cells
• Samples generated from tissue (including frozen patient biopsies)
• Clinical samples (PBMCs, BMMCs)

We provide detailed sample prep protocols and shipment instructions. For tissues, we can provide expert guidance on the best strategies for isolating cells.

Unfortunately, our CUT&RUN processes are not yet optimized for fixed tissues embedded in paraffin blocks (e.g. FFPE). Contact us to learn more about ongoing research efforts or alternate offerings to support this application.

For CUTANA CUT&RUN experiments, we recommend using 500,000 to 50,000 cells per reaction.*

Note that each reaction maps one target in one sample. Samples are unique cell populations, such as control vs. mutant or biological replicates.

If you have one cell sample, and you want to map three unique chromatin targets, you will be setting up three reactions – and thus require 1.5 million cells. We always suggest collecting 10-20% excess cells to account for sample loss.

* Our workflow is validated down to 10,000 cells for select targets. Success at low cell numbers depends on antibody performance, target abundance, and sample quality. There are additional risks assumed by going below the recommended cell numbers – such as low yields, increased adapter dimer contamination in libraries, increased background and/or sequencing depth – that may require additional optimization. Our scientists will help you weigh these risks during the experimental design phase of the project.

Controls are essential for overall project success, risk mitigation, and data interpretation. For this reason, EpiCypher’s CUT&RUN Services workflow includes a collection of quality control checks at each step of the process to help ensure you get the highest quality data possible.

Note that in our workflow, samples are unique cell populations, such as control vs. mutant or biological replicates. The term reaction is used to define the mapping of one target in one unique sample. For instance, if you have one cell sample and want to map three distinct chromatin targets, you will have a total of 3 CUT&RUN reactions.

Sample preparation

• Detailed methods for cell or nuclei collection and shipping instructions are provided to promote seamless sample handoff to our team.
• For tissues, our team can help develop a standard procedure for cell harvest.
• Upon arrival at EpiCypher, our team confirms cell number, viability, and integrity. Our scientists use these data to create a sample report that we review together to assess risk.
• Only samples that pass quality control metrics are included in experiments.

Control reactions including SNAP-CUTANA™ Spike-in Controls

• We set up three control reactions for each unique sample: H3K4me3 and H3K27me3 (positive controls) and IgG (negative controls). Example: If your project analyzes 5 unique PBMC samples, we set up three control reactions for each sample, or a total of 15 control reactions.
• Control reactions are used to validate sample quality, examine assay background, determine significant regions of enrichment above background (e.g. peaks), and confirm workflow success.
• Additionally, data from positive controls provide valuable biological context for chromatin regions associated with active (H3K4me3) and repressed (H3K27me3) genes.
• As an additional layer of protection, control reactions include our SNAP-CUTANA Spike-in Controls. SNAP-CUTANA Spike-ins comprise a defined, proprietary control that is spiked into reactions at the beginning of the experiment. The control is processed alongside biological samples through every step of the protocol, enabling direct monitoring and readout of experimental success. Learn more about our SNAP-CUTANA Spike-in Technology here.

E. coli Spike-in DNA

• All reactions are spiked with E. coli Spike-in DNA after targeted MNase cleavage to monitor library prep success.
• E. coli read counts are provided to customers, which can be used for sequencing normalization.

Pre-sequencing metrics

• EpiCypher scientists examine raw CUT&RUN-enriched DNA yields.
• Adjustments are made for low yields and/or low-abundant targets when going into library prep.
• Fragment distribution and concentration of sequencing libraries are determined using the Agilent TapeStation system. This is the best method to confirm CUT&RUN success before sequencing.

Post-sequencing metrics

• Libraries are sequenced to a depth that provides >3 million uniquely mapped reads. EpiCypher scientists use an optimized bioinformatic pipeline to align sequencing reads to the respective species genome and deliver high-quality data.
• We examine overall sequencing statistics, including unique alignment rates, duplication rates, and percent of reads assigned to E. coli and SNAP-CUTANA Spike-in controls, all of which help to determine sequencing success.
• Genomic and SNAP-CUTANA spike-in sequences are examined for control reactions (IgG, H3K4me3, H3K27me3) to ensure they produce the expected biological distribution with high specificity and signal-to-noise. This analysis lends confidence that the samples were high quality and the overall experiment was successful, enabling confident interpretation of the target(s) of interest.
• Peak calling pipelines are used to identify areas of significant target enrichment and assess overall signal-to-noise (Fraction of Reads in Peaks, or FRiP score).
• If multiple CUT&RUN experimental variables (e.g. antibodies, sample prep methods, etc.) are tested, the metrics above will define the optimal conditions for downstream analyses and future projects.

The EpiCypher Services Team has experience mapping various targets:

• Transcription factors (e.g. CTCF, FOXA1, p53, c-Jun)
• Chromatin remodeling enzymes (e.g. SMARCA2/BRM and SMARCA4/BRG1)
• Histone modifying enzymes and regulators (e.g. MLL1, EZH2, BRD4, Menin)
• Histone PTMs (methylation, acetylation, phosphorylation, ubiquitination)

As a result, we have a broad catalog of target-specific antibodies demonstrating exceptional performance in CUT&RUN. We are also highly skilled in sourcing and validating antibodies to new targets. Antibody screening is offered as part of our CUT&RUN Services and can be incorporated into your Statement of Work.

We work with every service partner to tailor our analysis to your needs. EpiCypher uses our bioinformatics pipeline – specifically optimized for CUT&RUN – to analyze sequencing data. At our data delivery meeting, we review preliminary data visualization at customer-identified target loci and all quality control checks (outlined above) to illustrate experimental success. We coordinate handoff of raw sequencing data, alignment files, bigWig files, and called peaks by SharePoint or Amazon Web Services. Files can be immediately used to visualize data, perform downstream analyses (e.g. differential enrichment), and derive biological insights in your lab. Specifically, we provide:

• Raw sequencing data in FASTQ files. EpiCypher performs paired-end sequencing, meaning each sequencing library has two FASTQ files (e.g. R1 and R2).
• Aligned, filtered reads in BAM files. The final BAM files contain uniquely aligned reads, with multimapping reads, exclusion list regions, and duplicate reads removed.
• Visualization-ready data in bigWig files. BigWig files are RPKM (Reads per Kilobase per Million mapped reads) normalized for visualization on genomic browsers (e.g. IGV).
• Signal enrichment in BED files, generated from peak calling.
• (As needed) Text file with motif analysis results, showing target-enriched sequences.

To see a sample data report, email services@epicypher.com

CUTANA CUT&RUN Service timelines vary, depending on sample prep quality, number of samples, and if antibody screening is required. Once samples are delivered to EpiCypher and have passed our quality control checks and risk assessment, it typically takes ~6 weeks to deliver data.

Ask our team about ongoing R&D efforts to profile DNA methylation, chromatin accessibility and more, as well as access precious clinical samples including liquid biopsies and formalin-fixed paraffin-embedded (FFPE) tissue.