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New Methods Enable Simultaneous Single-Cell Analysis of Chromatin, RNA, Proteins

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NEW YORK — A team led by researchers at the New York Genome Center has developed a pair of single-cell analysis techniques, one for profiling chromatin accessibility, mitochondrial DNA, and protein levels and the other for measuring chromatin accessibility, mRNA expression, and protein levels.

In a paper published this month in Nature Biotechnology, the researchers used the approaches, called ASAP (ATAC with select antigen profiling)-seq and Dogma-seq, to investigate changes at the level of chromatin, RNA, and proteins during hematopoietic differentiation and to observe changes in primary T cells due to various perturbations.

The development effort was inspired by previous work by Caleb Lareau, a science fellow at Stanford University and co-author on the study, said Peter Smibert, senior author on the study and VP of functional genomics at immune profiling firm Immunai, a role he began in February after leaving his position as director of the technology innovation lab at NYGC.

During a talk at the center, Lareau and his collaborator Leif Ludwig, a group leader at the Berlin Institute of Health, detailed a version of the single-cell ATAC-seq chromatin profiling method in which they deliberately preserved the cell's mitochondrial DNA for analysis.

Traditionally, Smibert noted, ATAC-seq users have tried to eliminate mitochondrial DNA from samples to preserve sequencing capacity for the chromatin analysis. "But these guys realized that if they could actually enrich and get high coverage of mitochondria, they could catalog the mutations that occur in the mitochondrial genome and use that as a way to look at relationships between cells," he said.

For this approach, they used whole cells as opposed to the isolated nuclei typically used for ATAC-seq experiments. Seeing this, Smibert and his colleagues realized that by using whole cells, they could similarly combine ATAC-seq chromatin analysis with measurements of mitochondrial DNA as well as cell surface and intracellular proteins, leading to the ASAP-seq method.

The researchers also developed a set of "linker" oligonucleotides that allowed them to use the extensive catalogs of oligo-conjugated antibodies developed for single-cell techniques like CITE-seq (BioLegend's TotalSeq reagents, for instance) with ATAC-seq protocols.

The accessibility of large numbers of oligo-tagged antibodies that can be made compatible with ATAC-seq means "that the barrier to entry for this kind of work is very low now," Smibert said.

In their recent study, the researchers measured as many as 242 proteins in the single cells they analyzed but could in theory go much higher, said Eleni Mimitou, first author on the paper.

"You could use the full 1,000 [oligo-conjugated antibodies] that BioLegend has made," she said. "It's all about how much money you want to spend in reagents and sequencing. We haven't reached a point yet where we've said, 'That's the limit. We can't go further.'"

Like Smibert, Mimitou recently joined Immunai, leaving her position as a senior research scientist at the NYGC in April to become associate director of single cell technologies at the company.

Mimitou noted that due to the fixation process, using whole cells as opposed to isolated nuclei appeared to have "a small impact on the complexity" of the chromatin fragments collected, with users reporting fewer unique fragments per cell. However, she said that this does not appear to "meaningfully impact" the ATAC-seq library quality.

As Smibert, Mimitou, and their colleagues were developing the ASAP-seq assay, 10x Genomics released its single-cell Multiome assays, which combines ATAC-seq with gene expression analysis. Having worked out how to combine ATAC-seq with protein measurements in developing the ASAP-seq assay, the researchers were able to add protein expression measurements to the Multiome approach relatively easily, Smibert said. With this, they were able to analyze single cells at the level of chromatin, RNA, and protein — the three components of the central dogma, which gave the assay its name, Dogma-seq.

"In a way it steals a bit of the novelty away from ASAP-seq," Smibert said, though he noted that given the expense of the 10x Genomics Multiome kit and sequencing more generally, he expected ASAP-seq would still see uptake from users who don't need the RNA data.

Mimitou added that Dogma-seq was less suited to analysis of intracellular proteins than ASAP-seq, given the difficulty of performing the multiple staining and washing steps required for protein measurements while preserving RNA for analysis.

"With all methods trying to do intracellular staining of proteins with RNA, there are really significant trade-offs in the RNA quality because RNA is super labile," Smibert said. "There will certainly be [RNA] signal there. We have internally done intracellular protein staining with Dogma-seq and it works, but the trade-off with RNA quality is pretty substantial."

In their recent study, the researchers used ASAP-seq to analyze chromatin, mtDNA, and protein to profile bone marrow cells, identifying 21 distinct clusters of cells while finding that protein-level data allowed them to distinguish between several cell types that were not well resolved by chromatin profiling. The authors noted that they expected the approach's ability to measure intracellular proteins would "spur the development" of antibody panels "targeting intracellular epitopes including signaling molecules, specific phosphor-epitopes, and [transcription factors]."

Smibert and Mimitou said they plan to continue developing single-cell multiomic techniques at New York City-based Immunai, which they joined as the firm is pushing into functional genomics to inform immune therapy development and applications.

"We're just trying to build a better microscope to try to see things we haven't been able to see before," Smibert said. "We have [at Immunai] the opportunity to do this at scale, to get access to very interesting patient cohorts and interesting experimental models, and [to] use these sorts of cutting edge tools to identify new targets for modulation and just understand the immune system better, both in normal functioning and dysfunction."

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