PROTEIN DETECTION BY SEQUENCING: TOWARDS A DEFINITIVE CELLULAR PHENOTYPE Methods that allow researchers to simultaneously sequence RNA and detect extracellular proteins in individual cells reveal new cell types and states associated with disease.

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DNA-tagged antibodies allow for detection of proteins with results read by sequencing. ELLA MARU STUDIO, INC.

PROTEIN DETECTION BY SEQUENCING: TOWARDS A DEFINITIVE CELLULAR PHENOTYPE

Methods that allow researchers to SIMULTANEOUSLY SEQUENCE RNA AND DETECT EXTRACELLULAR PROTEINS in individual cells reveal new cell types and states associated with disease.

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roteins are the workhorses A major advantage of comprehensive reference To validate CITE-seq, the of the cell, involved in CITE-seq and REAP-seq is maps of all 37 trillion cells in developers at the New York P almost every aspect of that, with an appropriate the human body. Genome Center and scientists structure and function. Protein antibody, they can detect Now, by providing details at NYU Lagone Health used expression patterns help define a protein of interest even if about both protein and RNA it to simultaneously profile a cell’s identity and state. the RNA that encodes it is in expression, CITE-seq and the transcriptome and 13 RNA transcripts are often low abundance. “CITE-seq REAP-seq allow researchers cell-surface proteins of used as a surrogate for protein gives us more confidence in to fill in the gaps left by flow human and mouse immune expression, but it has long been the presence of a marker of cytometry or single cell cells. They showed that understood that the relationship interest in a given cell type,” RNA-seq. CITE-seq not only correctly between abundance of proteins Rahman explains. “Advances in multimodal identified different immune and mRNA is not one-to- For decades, researchers single cell sequencing cell populations, it enhanced one. There are differences have used flow cytometry to methods are providing an the characterization of known caused by regulation of post- detect proteins in single cells unprecedented opportunity subtypes. Using CITE-seq transcriptional, translational and characterize cell types to look at biological they were able to identify and protein degradation. based on protein expression mechanisms of disease,” says subsets of natural killer “It has been eye-opening patterns. Flow cytometry Tom Maniatis, a renowned cells with different roles to see how much we might be relies on antibodies biochemist and biophysicist in regulating the immune missing by using conventional directed against proteins of leading Columbia University’s response in disease states sequencing,” concedes Adeeb interest. This means that to Precision Medicine Initiative that could not be detected Rahman, an immunologist obtain useful information and the New York Genome using single-cell RNA-seq at Icahn School of Medicine researchers need to know Center. Technologies that methods alone1. at Mount Sinai in New what they are looking for. combine single cell DNA or REAP-seq was first York. Rahman is developing RNA sequencing with methods employed to examine the innovative assays to monitor that provide details about the response of a T cell subset disease-relevant molecular “IT HAS BEEN regulation of gene expression, to a drug that activates the profiles and discover new EYE-OPENING TO protein expression, genetic immune system to attack biomarkers and drug targets. SEE HOW MUCH mutations, cell lineage, and and kill cancer cells in mice “In some cases, we have WE MIGHT even the spatiotemporal order by targeting a cell surface populations of cells that are all BE MISSING of molecular events not only protein. Differences between clearly expressing a particular BY USING allow the creation of a more the proteins and genes protein, but we may detect the CONVENTIONAL refined human cell atlas, but expressed in the treated and corresponding transcript in SEQUENCING.” offer new insights into cancer untreated cells provided only 5–10% of them,” evolution, neurodegeneration new insights into the drug’s he explains. and the immune system’s mechanism of action2. Rahman and others are In recent years, single response to disease. Since these studies were using techniques such as cell sequencing has become published, more than 200 CITE-seq (cellular indexing of the method of choice for Multimodal single cell oligo-conjugated antibodies transcriptomes and epitopes uncovering cell types sequencing in action have become available for by sequencing) and REAP- and states based on gene In CITE-seq and REAP-seq, CITE-seq and REAP-seq. seq (RNA expression and expression patterns. One of cells are incubated with These antibodies have been protein sequencing assay) to the advantages of single cell cell-surface antibodies used to assess immune investigate the relationship RNA (or DNA) sequencing is linked to oligonucleotide cell activation in patients between RNA transcripts that it is unbiased, it requires barcodes which tag the with advanced lung cancer and proteins, and identify no prior knowledge about protein’s identity, and a after immunotherapy3, new cell types and cell states the sample — RNA (or DNA) stretch of adenine bases that and to investigate associated with disease. Both from any cell type can be run serve as a starting point for immune dysregulations methods combine antibody- through a sequencer to obtain RNA sequencing (FIG). The at atherosclerotic sites oligonucleotides with existing its genomic or transcriptional technologies differ in the way associated with clinical single-cell RNA-sequencing profile. Advances in single- the DNA barcode binds to cerebrovascular events. (scRNA-seq) approaches to cell genomics are enabling the antibody. In both cases, When Rahman and measure the expression levels large scale projects such protein detection has been colleagues used CITE-seq of genes and cell-surface as the Human Cell Atlas shown to be as robust as by to examine the immune cell proteins in individual cells. Project, which aims to create flow cytometry. types in atherosclerotic

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lesions in stroke patients, blood contain the virus. active transcriptional tools are leading to a ‘second they found T cell subsets Moreover, HIV infected cells regulatory elements, wave’ of excitement around that were more activated, do not display any markers in nuclei obtained from single cell technologies. differentiated and that distinguish them from postmortem spinal cord cells “Now we can begin to really exhausted compared to uninfected cells, making to develop new treatments understand the complexity their blood counterparts. them practically impossible for spinal-cord disease of neurodegenerative Characterizing immune to detect and analyse. and injury. diseases, and the cell profiles with these ECCITE-seq can capture Rizvi is working closely contribution of multiple technologies could aid the the variability of T cell with bioinformaticians genes and variants to design of tailored therapies receptor sequences that and statisticians to disease processes in a and help predict a patient’s confer specificity for develop mathematical way that was not possible treatment response4. particular antigens. These approaches to harmonize before,” Maniatis adds. sequences are found at the Expanding the multimodal 5’ end of RNA molecules sequencing toolbox and are difficult to capture in “NOW WE CAN BEGIN TO REALLY Using the same concept scRNA-seq approaches that UNDERSTAND THE COMPLEXITY of DNA barcodes, CITE- profile the 3’ ends of RNAs. OF NEURODEGENERATIVE seq has been extended by Furthermore, this method DISEASES.” Peter Smibert and Neville can be expanded to capture Sanjana at the New York HIV RNA in the same cell. Genome Center to reveal “ECCITE-seq allows us to the data. “Multimodal Future outlook even more information tackle the heterogeneity, strategies enable us to Barcoding cellular content from single cells. ECCITE- rarity and the lack of marker make connections among is enabling researchers to seq (expanded CRISPR- in these cells, which is very simultaneously collected obtain multiple readouts compatible cellular indexing powerful,” she explains. measurements in any from single cells. As such, of transcriptomes and Ho and her team are cell type, and therefore they can characterize epitopes by sequencing), currently looking at cells in decipher key molecular cellular phenotypes can be used for the blood of HIV-infected events associated with the in more detail than by simultaneous detection of individuals during high- development, establishment using transcriptome the transcriptome, proteins, level viraemia and after and maintenance of neural measurements alone. clonotypes and CRISPR antiretroviral therapy to circuits,” he explains. These multi-omic readouts perturbations5. determine how infected When the team compares hold great potential to cells are able to continue the transcriptional advance translational dividing, leading to the clonal expression patterns from genomic science and “SINGLE CELL expansion of HIV-positive cells in cervical, thoracic and precision medicine. SEQUENCING cells. Understanding the lumbar regions of the spinal “We are also excited METHODS ARE features of these resistant cord, they see remarkable about the development PROVIDING AN clones may offer clues for differences. “Ultimately, of spatially resolved UNPRECEDENTED new therapies that target we want to carry out the transcriptomic OPPORTUNITY cells bearing the virus and, same type of analysis on approaches to place single TO LOOK AT effectively, cure HIV. the spinal cord of patients cell RNA sequencing data BIOLOGICAL Abbas Rizvi is a molecular with amyotrophic lateral in its anatomical context,” MECHANISMS OF neuroscientist working with sclerosis to understand the says Rizvi. This will allow DISEASE.” Maniatis and colleagues mechanisms underlying the identification of at Columbia University to motor neuron degeneration, cells that are vulnerable combine other single cell with the goal of identifying to neurodegeneration, “I love this technology,” sequencing technologies to novel therapeutic targets,” as well as to define the says Ya-Chi Ho, a molecular develop a comprehensive says Maniatis. features of cells that virologist at Yale School cell atlas of the human spinal Computational tools are surround them, and which of Medicine, who is trying cord. They are carrying out crucial to make biological are likely to contribute to to understand how HIV RNA-seq and ATAC-seq sense out of all the data that disease progression. persists in cells. HIV infected (assay for transposase- are being generated by new At present, single cell cells are very rare: less than accessible chromatin using sequencing technologies. multi-omics technologies 1% of T cells in peripheral sequencing), which identifies Rizvi observed that these are primarily used to

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identify correlations among CITE-SEQ & REAP-SEQ data types. But soon, by combining them with Barcoded cellular markers help simultaneously profile mRNAs experimental perturbations, and surface proteins from individual cells. it will be possible to explore causal relationships. Protein detection by Tagged antibody sequencing requires large,

5’ PCR HANDLE ANTIBODY BARCODE AAAAAAAAAAAAAAAAA(+A13)3’ pre-optimized panels of antibodies. At present there  Antibodies specific to cell surface proteins are AAAA seems to be no technical each tagged with an oligonucleotide which limit to the number of contains a barcode. Each barcode identifies a antibodies that can be particular cell-surface protein. used to detect extracellular proteins via CITE-seq. Mild permeabilization methods that allow antibodies to get inside cells may enable AAAA Antibody researchers to sequence barcode sequence Protein mRNA intracellular proteins in the AAAA future. “Such improvements would dramatically increase mRNAs the power and applicability AAAA of this method to other  Separate systems,” says Maniatis. AAAA tags from As CITE-seq and antibodies and  Antibody barcode sequences are ECCITE-seq antibodies isolate mRNA. captured, decoded, and quantitated, become increasingly as are corresponding mRNAs. These available, Rahman expects protein and mRNA profiles help identify these approaches will cell type, sample, species or lineage. become rapidly and widely adopted: “If you are already doing single cell  Applications RNA-seq, adding in CITE- seq is a pretty minimal investment for the amount of extra data you get.”

Changes REFERENCES in disease 1. Stoeckius, M. et al. Nat Methods 14, 865–868 (2017) state 2. Peterson, V. et al. Nat Biotechnol 35, 936–939(2017) Identify 3. Chin, V. T. et al. J Clin Onc 37, cell subtypes Predict response 15_suppl e20563 (2019). to treatment 4. Fernandez, D.M. et al. Preprint at bioRxiv https://doi. org/10.1101/721688 (2019) 5. Mimitou, E. P. et al. Nat Methods 16, 409–412 (2019) Assess response Develop to drug treatment tailored therapies

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