Simultaneous epitope and transcriptome measurement in single cells

Stoeckius, Marlon; Hafemeister, Christoph; Stephenson, William; Houck-Loomis, Brian; Chattopadhyay, Pratip K.; Swerdlow, Harold; Satija, Rahul; Smibert, Peter

doi:10.1038/nmeth.4380

Cited by 2,483 publications

(2,425 citation statements)

References 29 publications

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“…The Pro-Codes makes it possible to analyze millions of single cells with precise quantification of protein levels. Though the number of genes that can be analyzed by CyTOF is fewer than scRNA-seq, it should be feasible to expand the phenotyping space using oligo-labeled antibodies to detect the Pro-Codes and other proteins, and to deconvolute with single cell sequencing, as has been described (Peterson et al, 2017; Stoeckius et al, 2017). …”

Section: Discussionmentioning

confidence: 99%

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Wroblewska

Dhainaut

Ben-Zvi

et al. 2018

Cell

133

100

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Summary CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). ProCode-expressing vectors were introduced into cells and analyzed by CyTOF mass-cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells, and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single cell resolution.

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Section: Discussionmentioning

confidence: 99%

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Wroblewska

Dhainaut

Ben-Zvi

et al. 2018

Cell

133

100

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“…Another approach for increasing the antibody readouts per cell relies on conjugating antibodies with DNA oligonucleotides (barcodes), and a number of recent methods, including Abseq, cellular indexing of transcriptome and epitope by sequencing (CITE-seq), and RNA expression and protein sequencing (REAP-seq), have employed this strategy [48,53,54] (see Figure 1 and Table 1). With Abseq, single cells are incubated with barcoded antibodies recognizing cell-surface proteins; the method is limited to surface proteins.…”

Section: Antibody-based Methodsmentioning

confidence: 99%

Single cell protein analysis for systems biology

Levy

Slavov

2018

Essays in Biochemistry

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The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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“…Nevertheless, efforts have been made to quantify a few proteins of interest during scRNA-seq. Stoeckius et al [59] designed CITEseq (cellular indexing of transcriptomes and epitopes by sequencing). In this method, antibodies against multiple cell surface epitopes are labeled with oligonucleotides and then used to bind cells to be subject to scRNA-seq.…”

Section: Simultaneous Transcript and Protein Analysismentioning

confidence: 99%

Understanding the Biology and Pathogenesis of the Kidney by Single-Cell Transcriptomic Analysis

Song

Zhang

et al. 2018

Kidney Dis

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Background: Single-cell RNA-seq (scRNA-seq) has recently emerged as a revolutionary and powerful tool for biomedical research. However, there have been relatively few studies using scRNA-seq in the field of kidney study. Summary: scRNA-seq achieves gene expression profiling at single-cell resolution in contrast with the conventional methods of gene expression profiling, which are based on cell population and give averaged values of gene expression of the cells. Single-cell transcriptomic analysis is crucial because individual cells of the same type are highly heterogeneous in gene expression, which reflects the existence of subpopulations, different cellular states, or molecular dynamics, of the cells, and should be resolved for further insights. In addition, gene expression analysis of tissues or organs that usually comprise multiple cell types or subtypes results in data that are not fully applicable to any given cell type. scRNA-seq is capable of identifying all cell types and subtypes in a tissue, including those that are new or present in small quantity. With these unique capabilities, scRNA-seq has been used to dissect molecular processes in cell differentiation and to trace cell lineages in development. It is also used to analyze the cells in a lesion of disease to identify the cell types and molecular dynamics implicated in the injury. With continuous technical improvement, scRNA-seq has become extremely high throughput and cost effective, making it accessible to all laboratories. In the present review article, we provide an overall review of scRNA-seq concerning its history, improvements, and applications. In addition, we describe the available studies in which scRNA-seq was employed in the field of kidney research. Lastly, we discuss other potential uses of scRNA-seq for kidney research. Key Message: This review article provides general information on scRNA-seq and its various uses. Particularly, we summarize the studies in the field of kidney diseases in which scRNA-seq was used and discuss potential additional uses of scRNA-seq for kidney research.

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Simultaneous epitope and transcriptome measurement in single cells

Cited by 2,483 publications

References 29 publications

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Single cell protein analysis for systems biology

Understanding the Biology and Pathogenesis of the Kidney by Single-Cell Transcriptomic Analysis

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