Bringing epigenomic profiling to the single-cell biology stage

By their nature, researchers are big thinkers and driven to innovate. However, sometimes the current methods available simply can't keep up. Having tools that enable you to develop new techniques is crucial for you to move your research into new arenas not previously possible.

The ICELL8 Single-Cell System was designed with this type of openness and flexibility in mind, and many researchers are taking advantage of these features. For example, Dr. Steven Henikoff's laboratory (Fred Hutchinson Cancer Research) used the platform to develop a new technique for epigenetics—CUT&Tag (Cleavage Under Targets and Tagmentation)—that allows chromatin mapping not only at high resolution but at the single-cell level (Kaya-Okur et al. 2019).

Going beyond ChIP-seq

Epigenetics focuses on understanding how the structure of DNA, histone modifications, and DNA methylation can drive drastically different gene expression and phenotypic changes for the same genotype (Deichmann 2016). While ChIP-seq has been an invaluable tool for epigenetic analyses, its utility is limited due to a large sample-input requirement and data analysis that is complicated by a poor signal-to-noise ratio.

Developing an experimental strategy with low background was a crucial step to allow the profiling of fewer cells. In 2017, Skene and Henikoff accomplished this with an enzyme-tethering technique termed CUT&RUN (Cleavage Under Targets and Release Using Nuclease; Skene and Henikoff 2017) which uses a protein A-monococcal nuclease (protein A-MNase) fusion to specifically cleave DNA on both sides of transcription-factor-DNA (TF-DNA) complexes bound by antibodies in situ rather than relying on crosslinking strategies like other ChIP methods.

Getting to the single-cell level

Although the CUT&RUN method is amenable to automation and can work for samples with low numbers of starting cells (100–1,000), Henikoff's group saw room for improvement. Specifically, they wanted to eliminate the need for DNA end polishing and adapter ligation to prepare sequencing libraries from the retrieved TF-DNA complexes. They accomplished this by replacing the protein A-Mnase fusion with protein A fused to a hyperactive Tn5 transposase (pA-Tn5). Thus, the CUT&Tag method was developed. When activated this pA-Tn5 integrates its preloaded sequencing adapters at both ends of the TF-DNA complexes to create PCR-enrichment-ready fragments, improving assay signal range 20-fold compared to bulk ChIP-seq experiments.

Overview of the CUT&Tag method and workflow on the ICELL8 Single-Cell System. Panel A. Schematic depicting the CUT&Tag process. The target protein (blue) is bound by the primary antibody (green), which is then bound by a secondary antibody (orange). An adapter (red)-loaded Tn5 transposase conjugated to protein A (grey) then binds the secondary antibody, tethering it and promoting the incorporation of adapters at the target site. Fragments are then enriched by PCR. Panel B. Workflow of single-cell CUT&Tag as performed on our ICELL8 Single-Cell System. (Figure by Kaya-Okur et al. 2019, used under CC-BY-NC-ND 4.0)

The authors reasoned that the CUT&Tag's sensitivity would allow single-cell profiling (scCUT&Tag). Since the protocol requires permeabilization of the cells, rendering them extremely fragile, a system that isolates single cells as gently as possible was necessary. Our automated ICELL8 platform was used to aliquot these permeabilized cells into a 5,184-well chip following tagmentation. Furthermore, the integrated imaging capabilities of the system allowed visual inspection of the wells so that only wells containing one cell would be processed for PCR enrichment using their own customized reagent setup. Last, but certainly not least, the researchers took advantage of a particular reagent dispensing script on the ICELL8 system to trace that data back to particular cells by dispensing 72 i5 and 72 i7 indices in such a way that each cell received a unique index combination. In proof of concept experiments, the authors found that the data from this scCUT&Tag setup corresponded well with chromatin profiling from their bulk experiments (Figure 6 in publication).

Sequence better, faster, and deeper

Our ICELL8 system provides a flexible, adaptable platform for assay development and optimization. In this article, we provided a glimpse of how Dr. Henikoff's laboratory used our platform to develop their novel scCUT&Tag technology, but they are far from alone. Other researchers have used the ICELL8 platform to automate and perform single-cell sequencing. Stay tuned for additional posts telling the stories of these exciting applications.

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References

Deichmann, U. Epigenetics: The origins and evolution of a fashionable topic. Dev. Biol. 416, 249–254 (2016).

Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. bioRxiv 568915 (2019).

Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, (2017).