In human cells, we have found that the Hia5:nuclei ratio detailed in the Fiber-seq manual is ideal for obtaining optimal 6mA labeling (~6% labeling across the entire human genome). However, when working with a new species or unique conditions, it may be worthwhile to run an optimization experiment to experimentally determine this ratio in your sample type, as outlined below:
Recommended Workflow
1. Perform a 3-Point Cell Titration
To determine the optimal conditions for 6mA labeling, set up three parallel Fiber-seq reactions using varying nuclei inputs:
500,000 nuclei (or 0.5× the standard input based on your cell’s genome—see more on standard inputs here)
1,000,000 nuclei (or the standard input based on your cell’s genome)
2,000,000 nuclei (or 2× the standard input based on your cell’s genome)
This range allows you to assess how input quantity impacts labeling efficiency and overall data quality.
2. Run Fiber-seq Reactions
Process each condition independently through the full Fiber-seq workflow, including library prep. Be sure to keep all other variables consistent across reactions to ensure that differences in output are attributable to input amount.
3. Perform Shallow Sequencing
Sequence each sample at approximately ~1× coverage. This level of sequencing is sufficient to evaluate 6mA labeling efficiency without committing to a full-depth run. For more on QC sequencing, see our section on it here.
4. Evaluate % 6mA Labeling
Analyze the sequencing data to determine the percentage of 6mA incorporation for each condition. This metric serves as a key indicator of labeling efficiency.
Identify the input condition that provides optimal labeling (e.g., strong signal without over-labeling or signal saturation—in human cells, EpiCypher finds that 6% labeling achieves these goals).
Consider overall data quality in addition to % 6mA when selecting conditions.
5. Proceed with Final Experiment
Once the optimal input amount has been determined, use those conditions for your full-scale Fiber-seq experiment.
Additional Considerations
Limited or precious samples:
If your sample availability is constrained, consider performing the titration using a closely related cell type. This can provide a useful approximation for optimizing conditions while preserving your primary material.Enzyme titration option:
In addition to varying cell input, you may also perform a titration of the Hia5 methyltransferase concentration:0.5× standard concentration
1× standard concentration
2× standard concentration
This can help fine-tune labeling efficiency, particularly if cell input is fixed or limited.
By incorporating a brief titration step into your workflow, you can significantly improve the reliability and interpretability of your Fiber-seq results.