Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Gerlach, Jan P.; Buggenum, Jessie A.G.L. van; Tanis, Sabine E.J.; Hogeweg, Mark; Heuts, Branco M. H.; Muraro, Mauro J.; Elze, Lisa; Rivello, Francesca; Rakszewska, Agata; Oudenaarden, Alexander van; Huck, Wilhelm T. S.; Stunnenberg, Hendrik G.; Mulder, Klaas W.

doi:10.1101/356329

Cited by 8 publications

(15 citation statements)

References 35 publications

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“…Recently, two similar approaches, CITE-seq (9) and REAP-seq (10), have been described to measure protein expression using oligo-conjugated antibodies in parallel with scRNA-seq data. Furthermore, other applications are currently being developed to integrate the growing portfolio of single-cell omics technologies (35,36). A fundamental difference with the approach described in this study is that these technologies all rely on whole-transcriptome data, providing a high-level cross-sectional representation of all polyA transcripts in the cell.…”

Section: Discussionmentioning

confidence: 99%

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Trzupek

Dunstan

Cutler

et al. 2019

Preprint

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The transcriptomic and proteomic characterisation of CD4 + T cells at the single-cell level has been performed traditionally by two largely exclusive types of technologies: single cell RNAsequencing (scRNA-seq) technologies and antibody-based cytometry. Here we demonstrate that the simultaneous targeted quantification of mRNA and protein expression in single-cells provides a high-resolution map of human primary CD4 + T cells, and reveals precise trajectories of Th1, Th17 and regulatory T-cell differentiation in blood and tissue. We report modest correlation between mRNA and protein in primary CD4 + T cells at the single-cell level, highlighting the value of protein expression data to characterise cell effector function. This transcriptomic and proteomic hybrid technology provides a massively-parallel, cost-effective, solution to dissect the heterogeneity of immune cell populations and to immunophenotype rare and potentially pathogenic immune subsets, and could have important clinical applications to redefine differentiation and activation of circulating and tissue-resident human immune cells.

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Section: Discussionmentioning

confidence: 99%

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Trzupek

Dunstan

Cutler

et al. 2019

Preprint

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“…genetic or chemical perturbations. We propose that the approach can be used for the analysis of transcript isoform usage and protein expression as explored in Gerlach et al 13 and will be valuable for analyses of the relationship between mRNA and protein expression variation. The proximity-based protein assays are well suited to detect post-translational modifications 33 and can be scaled up via a sequencing readout 34 , allowing measurement of still greater numbers of proteins and protein modifications in individual cells.…”

Section: Discussionmentioning

confidence: 99%

“…A number of approaches are emerging to simultaneously measure both mRNA and protein at single cell resolution. These include measurement of cell surface proteins and mRNA in droplet microfluidic-based analysis platforms 7,8 , targeted mRNA and protein detection [9][10][11][12] , and mRNA and targeted protein measurement for a limited number of cellular proteins (n=6) in fixed cells 13 . We describe an approach, herein called single-cell protein and RNA co-profiling (SPARC), which enables measurement of global mRNA and high multiplex, targeted cellular proteins in single cells ( Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Combined mRNA and protein single cell analysis in a dynamic cellular system using SPARC

Reimegård

Danielsson

Tarbier

et al. 2019

Preprint

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Combined measurements of mRNA and protein expression in single cells enables in-depth analysis of cellular states. We present single-cell protein and RNA co-profiling (SPARC), an approach to simultaneously measure global mRNA and large sets of intracellular protein in individual cells. Using SPARC, we show that mRNA expression fails to accurately reflect protein abundance at the time of measurement in human embryonic stem cells, although the direction of changes of mRNA and protein expression are in agreement during cellular differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do the corresponding transcripts. We further show that changes of the balance between protein and mRNA expression levels can be applied to infer expression kinetic trajectories, revealing future states of individual cells. Finally, we highlight that mRNA expression may be more varied among cells than levels of the corresponding proteins. Overall, our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. Accordingly, SPARC can offer valuable insights in gene expression programs of single cells.

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“…Single‐cell RNA and immuno‐detection (RAID) was devised to enable the combined measurement of intracellular protein and the transcriptome. [ 89 ] As demonstrated in Figure 9B, RNA sequences containing barcodes and poly (A) are designed and produced by in vitro transcription and are conjugated to antibodies by a Diels–Alder reaction. Cells carry out fixation and immunostaining with antibody‐RNA complexes to label intracellular phospho‐protein in situ, and are sorted into a 384‐well plate containing barcode primers.…”

Section: From Transcriptome To Multi‐omicsmentioning

confidence: 99%

Single‐Cell Sequencing Methodologies: From Transcriptome to Multi‐Dimensional Measurement

et al. 2021

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Cells are the basic building blocks of biological systems, with inherent unique molecular features and development trajectories. The study of single cells facilitates in‐depth understanding of cellular diversity, disease processes, and organization of multicellular organisms. Single‐cell RNA sequencing (scRNA‐seq) technologies have become essential tools for the interrogation of gene expression patterns and the dynamics of single cells, allowing cellular heterogeneity to be dissected at unprecedented resolution. Nevertheless, measuring at only transcriptome level or 1D is incomplete; the cellular heterogeneity reflects in multiple dimensions, including the genome, epigenome, transcriptome, spatial, and even temporal dimensions. Hence, integrative single cell analysis is highly desired. In addition, the way to interpret sequencing data by virtue of bioinformatic tools also exerts critical roles in revealing differential gene expression. Here, a comprehensive review that summarizes the cutting‐edge single‐cell transcriptome sequencing methodologies, including scRNA‐seq, spatial and temporal transcriptome profiling, multi‐omics sequencing and computational methods developed for scRNA‐seq data analysis is provided. Finally, the challenges and perspectives of this field are discussed.

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Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Abstract: 1 9 7

Cited by 8 publications

References 35 publications

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Combined mRNA and protein single cell analysis in a dynamic cellular system using SPARC

Single‐Cell Sequencing Methodologies: From Transcriptome to Multi‐Dimensional Measurement

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