Using SNAP-CUTANA Spike-ins to troubleshoot workflows
It may be unclear from genomic tracks alone if a reaction issue has occurred. Pairing SNAP-CUTANA™ Spike-in Controls with EpiCypher’s robust control antibodies enables users to flag failed reactions AND identify potential causes for troubleshooting. Together, these controls allow researchers to be confident in their experimental results.
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.
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 & IgG control reactions to validate workflows for three independently prepared mouse B cell samples.
- Samples 1 & 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 & 2 generated expected tracks for H3K4me3 & H3K27me3, while Sample 3 profiles had poor S:N (Figure 2).
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 & 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.
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).
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 Table 1 (below).
Table 1. Troubleshooting using SNAP-CUTANA Spike-ins
| Results | Causes and troubleshooting approaches |
|---|---|
K-MetStat spike-in data: • High target specificity • High S:N • Genomic data: Poor S:N |
pAG-MNase cleavage and wash conditions are optimized. Control antibodies are performing as expected. Problems may include: ⚠ Low numbers of cells • Optimize assay with 500,000 cells before decreasing input • If using nuclei, adherent cells, cross-linked cells, tissues, or cryopreserved samples, see this section for modifications ⚠ Poor sample prep • Optimize Digitonin permeabilization of cells • 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; consider using the CUTANA CUT&RUN Library Prep Kit ⚠ 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 • For concerns about control antibody performance, email us at techsupport@epicypher.com |
