What samples and cell types are compatible with Fiber-seq?

Fiber-seq is compatible with a wide range of inputs and has been successfully applied to human cell lines, primary cells, plants, yeast, and non-model organisms. The key requirement is that DNA be free of endogenous N⁶-methyladenosine as Fiber-seq relies on detecting 6mA, introduced during the assay by Hia5, to reveal open chromatin profiles. Thus, species that endogenously methylate their DNA (like most bacteria) are not compatible with Fiber-Seq.

Fresh nuclei provide the best results, though flash-frozen and cross-linked samples are also compatible, with minor labeling differences. Sample quality and genome size are important factors that influence input requirements and overall data quality.

Sample Input

• Recommended: 1,000,000 fresh nuclei* per reaction. To account for sample loss, start with 2M cells with the goal of retaining 1M nuclei. If you are using non-human cells, the target number of nuclei may be different.

• Flash-frozen and crosslinked nuclei are compatible (with slight labeling differences)

• Harvest ~2M cells* per reaction with 10% extra if possible to account for sample loss

Currently, inputs <1M nuclei are not validated. Scaling (ie decreasing Hia5 concentration for lower cell/nuclei inputs) is theoretically possible but not recommended at this time.

Genome Size

There is an inverse relationship between genome size and number of nuclei required for high-quality data. For organisms with smaller genomes, you'll need more nuclei to match the total DNA content of 1,000,000 human nuclei (the standard input the EpiCypher protocol is based on). This ensures consistent labeling and yields enough DNA for long-read sequencing.

Recommendation: Keep the reaction volume and enzyme concentration the same, but adjust the number of target nuclei to match total DNA input of 1,000,000 human nuclei.

Below is a table of common research organisms and the recommended number of nuclei to use per Fiber-seq reaction.

*human cells assumed. If using non-human cells, refer to the “Genome Size” section of this article.