---
title: "Troubleshoot workflows using SNAP-CUTANA™ Spike-ins"
slug: "using-snap-cutana-spike-ins-to-troubleshoot-workflows-cut-and-tag"
updated: 2024-10-01T17:15:03Z
published: 2024-10-01T17:15:03Z
canonical: "support.epicypher.com/using-snap-cutana-spike-ins-to-troubleshoot-workflows-cut-and-tag"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://support.epicypher.com/llms.txt
> Use this file to discover all available pages before exploring further.

# Troubleshoot workflows using SNAP-CUTANA™ Spike-ins

Here, we will discuss how to use SNAP-CUTANA Spike-in results to troubleshoot problematic workflows. To learn how to apply SNAP-CUTANA Spike-ins to confirm workflow success, see [this article](/v1/docs/using-snap-cutana-spike-in-controls-to-determine-cutrun-success). Note that while the data shown here is from CUT&RUN, these principles can be applied in all CUTANA assays.

## Example Data from a CUT&RUN Experiment

**Figures 1** and **2** demonstrate the use of K-MetStat Spike-in data for troubleshooting. In **Figure 1** we used spike-in data from H3K4me3, H3K27me3, and IgG control reactions to validate CUT&RUN workflows for three independently prepared mouse B cell samples. Key takeaways:

- Samples 1 and 2 showed expected results from control reactions, while Sample 3 displayed low signal-to-noise (S:N) and high off-target PTM recovery (**Figure 1**).
- Genomic profiles agreed with spike-ins: Samples 1 and 2 generated expected tracks for H3K4me3 & H3K27me3, while Sample 3 profiles had poor S:N (**Figure 2**).

To troubleshoot Sample 3 reactions, we considered the following:

- All reactions were performed in parallel using the same antibodies and reagents. However, only Sample 3 reactions had problems with background.
- Sample 3 showed poor S:N in both genomic profiles and K-MetStat Panel data.
- Sample 3 generated poor profiles across multiple targets.

Combined, these results suggested problems with sample prep vs. a complete workflow failure. We subsequently reviewed Sample 3 processing methods, revealing that the number of cells used per reaction was much lower than intended. For other troubleshooting tactics using the K-MetStat Panel, see the [**Table (below)**](/v1/docs/using-snap-cutana-spike-ins-to-troubleshoot-workflows#troubleshooting-cutana-assays-using-snapcutana-spikein-data).

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/2024.09.05 SNAP K-MetHeatmap pass_fail@2x.png)

**Figure 1.** K-MetStat Spike-ins validate workflows and flag poor samples in CUTANA CUT&RUN experiments. Spike-in data for H3K4me3 positive control reactions is shown for three independently prepared mouse B cell samples (10,000 cells each; protocol optimization experiment with a multi-lab consortium). Samples 1 and 2 show expected results, while Sample 3 was flagged for recovery of off-target PTMs and low signal-to-noise. Representative data from one IgG reaction is shown as a negative control.

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/KMetStat Flags bad samples updated@2x.png)

**Figure 2.** Genomic tracks featuring data in **Figure 1**. CUT&RUN was used to map IgG (negative control), H3K4me3 (positive control) and H3K27me3 in three independently prepared mouse B cell samples (10,000 cells each; protocol optimization experiment with a multi-lab consortium). A representative 400 kb region is shown. Samples 1 and 2 show consistent peaks, while Sample 3 displays low S:N (red).

  

## **SNAP-CUTANA Spike-in Troubleshooting Table**

| **Results** | **Causes and troubleshooting approaches** |
| --- | --- |
| K-MetStat spike-in data: - High target specificity - High S:N Genomic data: - Poor S:N | pAG-MNase/pAG-Tn5 cleavage and wash conditions are optimized. Control antibodies are performing as expected. Problems may include: ⚠ Low numbers of cells - Optimize assay with recommended number input (CUT&RUN: 500,000 cells, CUT&Tag: 100,000 nuclei) - For nuclei, adherent cells, cross-linked cells, tissues, or cryopreserved samples, see Sample Prep section for modifications ⚠ Poor sample prep - If using whole cells, optimize Digitonin permeabilization - Confirm sample integrity and bead binding - Avoid ConA bead clumping and dry out during assay ⚠ Experimental target requires different processing conditions - Ensure target is present and localized to chromatin - If using frozen cells, try freshly isolated cells - Test native vs. lightly cross-linked conditions |
| K-MetStat spike-in data: - Nonspecific PTM recovery - Poor S:N Genomic data: - Poor S:N | ⚠ Indicates a fundamental failure in the workflow - Carefully re-read the protocol and important notes - Ensure buffers are prepared fresh on day of use - Ensure ConA beads are in good condition (e.g. never frozen) - Make sure correct parameters are used in indexing PCR ⚠ Low numbers of cells and/or poor sample prep - Optimize following the guidelines above |
| K-MetStat spike-in data: - Nonspecific PTM recovery - S:N may vary Genomic data: - High S:N | ⚠ Indicates cross-reactive control antibodies - Examine potential contamination of control reactions with antibodies to other targets - Ensure buffers are prepared fresh on day of use - Change pipette tips after each reagent addition to avoid cross-contamination - [Contact us](https://www.epicypher.com/contact-us/) for concerns about control antibody performance |

---