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. 2019 Feb 6;9(1):1469.
doi: 10.1038/s41598-018-37977-7.

"V体育平台登录" Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Jan P Gerlach  1   2 , Jessie A G van Buggenum  1 , Sabine E J Tanis  1 , Mark Hogeweg  1 , Branco M H Heuts  1 , Mauro J Muraro  3 , Lisa Elze  1 , Francesca Rivello  4 , Agata Rakszewska  4 , Alexander van Oudenaarden  3 , Wilhelm T S Huck  4 , Hendrik G Stunnenberg  2 , Klaas W Mulder  5
Affiliations

"VSports手机版" Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Jan P Gerlach et al. Sci Rep. .

Abstract

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output VSports手机版. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level. .

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V体育官网入口 - Conflict of interest statement

M. J. M. is the CEO of Single Cell Discoveries, a company that provides a single-cell sequencing service. The other authors declare no competing interests V体育安卓版.

V体育官网入口 - Figures

Figure 1
Figure 1
Schematic overview of the RAID workflow. Cells are crosslinked, permeabilized and stained with Antibody RNA-Barcode Conjugates (ARCs) in suspension. Hereafter, cells are sorted into 384-wells plates where crosslinking is reversed and cDNA synthesis is performed using CELseq2 compatible dTV primers. Finally, single-cell samples are pooled and sequencing library preparation is performed using an adapted CELseq2 protocol to incorporate ARC measurements in the library.
Figure 2
Figure 2
Combined single-cell transcriptomics and antibody-barcode conjugate (ARC) detection from unfixed keratinocytes. (A) Boxplot showing mass spectrometry based analysis of the EGFR to ITGA6 ratio from bulk samples. AG1478 induced differentiation leads to a reduction of the EGFR to ITGA6 ratio. Experiment was performed in triplicate. (B,C) Single-cell analysis of unfixed keratinocytes stained with ARCs for EGFR and ITGA6. Cell were untreated or treated with the EGFR inhibitor AG1478 to induce differentiation. (B) Scatterplot shows the total number of UMI counts that were detected per cell from the transcriptome and from ARCs. Cells that passed the count thresholds for the mRNA (10,000) and ARCs (2,750) are indicated in green. (C) tSNE visualization using principal component 1 to 8 of the variable transcripts. Untreated and AG1478 treated cells form distinct clusters. Different batches of cells are color coded. (D) Featureplot showing the ratio of the UMI counts of EGFR-ARC and ITGA6-ARC for each cell projected on the tSNE coordinates from (C). (E) Ratio of the UMI counts of EGFR-ARC and ITGA6-ARC for control and AG1478 treated cells represented in violin plot.
Figure 3
Figure 3
Effect of the RAID procedure on single-cell transcriptomics quality. Comparison of single-cell transcriptomics quality between unfixed cells and cells that passed the complete RAID procedure including DSP/SPDP based fixation and mock ARC staining. Comparison is based on 40,000 sampled UMI counts per cell. (A) Boxplots showing a mild reduction of the gene complexity after the complete RAID procedure. (B) Scatterplot showing the average expression of genes in unfixed cells and RAID cells. Red line represents the x = y function as a visual aid. (C) Scatterplot showing the relation between the gene detection rate and the average gene expression in unfixed (blue dots) and RAID cells (red circles). (D) Scatterplot showing the relation between the coefficient of variation and the average gene expression in unfixed (blue dots) and RAID cells (red dots). R denotes Pearson Correlation.
Figure 4
Figure 4
RAID analysis shows association of FAK phosphorylation with the expression of stem cell marks. RAID was performed with ARC-based quantification of six antibodies from fixed keratinocytes. Cells were untreated or treated with the EGFR inhibitor AG1478 to induce differentiation. (A) tSNE visualization using principal component 1 to 8 of the variable transcripts. Untreated and AG1478 treated cells form distinct clusters. Different batches of cells are color coded. (B) Violin plots representing the ARC UMI counts for the indicated antibodies, based on a total of 400 sampled ARC counts per cell. (C) Featureplots showing data from B, projected on the tSNE embeddings from (A). (D–G) From the AG1478 treated cells, 50 cells were selected with the highest and lowest pFAK-ARC counts (pFAK-high and pFAK-low, respectively) and analyzed based on proteomic and transcriptomic features. (D) Histograms showing the distribution of UMI counts for the indicated ARCs in pFAK-high (orange) and pFAK-low cells (grey). (E) Boxplots showing the differentiation scores of pFAK-high and pFAK-low cells. (F) Boxplots showing the stem cell scores of pFAK-high and pFAK-low cells. The stem cell score is significantly higher in pFAK-high cells based on two tailed t-test (p = 0.0023). (G) Histograms showing distribution of UMI counts of the indicated integrin substrates per ten cells after 1000x random sampling from pFAK-high (orange), pFAK-low cells (grey) and total AG1478 treated cells (blue dashed line). Transcript UMI counts are based on 4,500 sampled UMI counts per single cell.
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References

    1. Regev A, et al. The Human Cell Atlas. eLife. 2017;6:e27041. doi: 10.7554/eLife.27041. - DOI - PMC - PubMed
    1. Macosko EZ, et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015;161(5):1202–1214. doi: 10.1016/j.cell.2015.05.002. - V体育官网 - DOI - PMC - PubMed
    1. Klein AM, et al. Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell. 2015;161(5):1187–1201. doi: 10.1016/j.cell.2015.04.044. - DOI - PMC - PubMed
    1. Jaitin DA, et al. Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types. Science. 2014;343(6172):776–779. doi: 10.1126/science.1247651. - "VSports最新版本" DOI - PMC - PubMed
    1. Stoeckius M, et al. Simultaneous epitope and transcriptome measurement in single cells. Nature Methods. 2017;14:865. doi: 10.1038/nmeth.4380. - DOI - PMC - PubMed

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