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High-Throughput Single-Cell Transcriptomics Assay Facilitates Compound Screens

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NEW YORK – A new method offers the possibility to assay the unique transcriptome response of millions of single cells to thousands of chemical perturbations, according to its developers.

Sci-Plex, developed by researchers in the labs of Jay Shendure and Cole Trapnell at the University of Washington, combines single-cell transcriptome sequencing with nuclear hashing, or labeling, to create a high-throughput screen with potential applications in drug discovery and basic research.

"Sci-Plex is just a way to look at lots of specimens at once," Trapnell said, and it builds on methods the two labs have been developing for several years. "Previously we could [analyze] millions of cells from a smaller number of samples. Now we can do millions of cells from thousands or tens of thousands of samples."

In a proof-of-concept study published today in Science, the researchers demonstrated the applicability of sci-Plex for high-throughput screening in drug discovery. The researchers exposed hundreds of thousands of cells from three cancer cell lines to 188 drug compounds in multiple doses, resulting in approximately 650,000 single-cell transcriptome profiles.

In addition to drug discovery, the method has potential applications in basic science, "where you want to look at a lot of different conditions and the inherent single-cell heterogeneity in the system," Trapnell said. "In principle, it could be combined with other techniques for perturbing cells or engineering cells," he added.

"You can get a sort of global view of the cellular responses," first author Sanjay Srivatsan said in a statement. "We think it's going to be really powerful to categorize drugs, for example, and say what their mechanism is."

Sci-Plex is not the first method of nuclear hashing, which labels different samples prior to transcriptome analysis, and it's not the first method to apply transcriptomics to find a middle ground between coarse cell viability assays and granular molecular target-focused assays.

For nuclear hashing, other researchers have used oligonucleotide-barcoded antibodies to label cells, for example in cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), or in multiplexing using lipid-tagged indices for single-cell and single-nucleus sequencing (Multi-Seq.)

Also, several groups, including researchers at the pharma giant Novartis, have developed bulk methods for transcriptome profiling in drug discovery. In 2018, researchers from the Novartis Institutes for Biomedical Research (NIBR), for example, published a paper on Drug-seq, which uses barcodes to track transcripts through the wells and plates used in screening.

"We now understand that the transcriptome can serve as a high-dimensional phenotype," Robert Ihry, a researcher at NIBR and an author on the Drug-seq paper, said in an email. He added that these types of transcriptomic assays are helping Novartis shift from a target-focused view of drug discovery to a "compound-centric view that attempts to capture biological activity holistically."

In addition to nuclear hashing, sci-Plex incorporates a single-cell transcriptome sequencing method developed by Trapnell and Shendure's teams, called single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3), which they described in a paper published in February.

Sci-RNA-seq3 uses three rounds of indexing using custom reverse transcriptase primers. "Every cDNA made is tagged with the well in which it was made," Trapnell explained. In each round, the cells are mixed and redistributed to a new plate, with transcripts receiving a new index. After RNA-seq, "any time you see two reads that have the same indices, based on probability, you can expect they came from the same cell," Trapnell said.

While the combinatorial indices track which cell the transcripts came from, the nuclear hashing helps track which experimental conditions the cell was exposed to. Sci-Plex's hashing method is simple: the oligos stick to nuclei separated from the rest of the cell. Trapnell admitted he's not entirely sure why it works — he speculates it is due to a "nonspecific electrostatic interaction between DNA and nuclear proteins — but it's enough that it does.

This method is different from some other methods that require physical isolation of cells, such as the 10x Genomics platform, Trapnell noted. Sci-plex is really cheap, though — he estimated that library construction costs were less than a cent per cell, on a per-condition basis — and the hashing oligos can be ordered directly from a supplier, such as Integrated DNA Technologies. The method becomes more useful the more samples one is looking at.

"If you're looking at less than 10 or 20 specimens, different samples, it's probably not needed," Trapnell said. "Once you get to more than, say, 30, 40, 50, samples, it starts to kick in."

Sci-Plex has only been demonstrated using sci-RNA-seq, but theoretically, it could work with another single-cell RNA-seq method, Trapnell said.

In addition to drug screens, Trapnell said the method could be useful in studying clinically relevant genetic variants and their impact on a cell's response to different drugs, for example. "Physicians also give many people the same handful of drugs, and they work for some people and not for others," Trapnell added. "Potentially, sci-Plex could help us better understand why that is."

Trapnell declined to discuss the intellectual property situation surrounding sci-Plex, but in the paper, the researchers wrote that "one or more embodiments of one or more patents and patent applications filed by Illumina and University of Washington may encompass the methods, reagents, and data disclosed in this manuscript." Also, Illumina Senior Scientist Fan Zhang is an author on the paper.

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