Single Cell REAP-Seq Service
Single Cell REAP-Seq (RNA expression and protein sequencing assay) is a technique for measuring the levels of gene and protein expression in single cells using DNA-labeled antibodies and droplet microfluidic.
Introduction to Single Cell REAP-Seq
REAP-seq permits the simultaneous measurement of proteins and mRNAs in individual cells. Rather than fluorophores, antibodies conjugated to DNA barcodes are used to label cells via techniques similar to the flow cytometry standard. This eliminates the restrictions imposed by the spectral overlap of fluorescent labels or the number of stable isotopes available in flow and mass cytometry.
Fig.1 Schematic of single cell REAP-seq. (Choi, 2020)
Published Data
| Paper Title | Multiplexed quantification of proteins and transcripts in single cells |
| Journal | Nature Biotechnology |
| Published | 2017 |
| Abstract | Using DNA-labeled antibodies and droplet microfluidics, they present a method for measuring gene and protein expression in single cells. Utilizing REAP-seq, they quantified proteins using 82 barcoded antibodies and more than 20,000 genes in a single workflow. REAP-seq was utilized to evaluate the costimulatory effects of a CD27 agonist on human CD8+ lymphocytes and to identify and characterize an unidentified cell type. |
| Result |
They analyzed cells treated with aCD27 using REAP-seq. The untreated and aCD27-treated samples from each donor were combined into a digital gene or protein expression matrix and unsupervised clustering was performed. To visualize the co-localization of cells within t-SNE plots, cells were color-coded according to their treatment conditions (Fig. 2a). There were 74 overlapping differentially expressed genes among the three donors (Fig. 2b). When cells were treated with aCD27, REAP-seq protein analysis identified 16 differentially expressed proteins that overlapped among the three donors (Fig. 2c). Expression of the nave T-cell marker CD45RA decreased, while expression of the effector memory T-cell marker CD45RO increased (Fig. 2d). They discovered an increase in ICOS protein expression but not at the transcriptional level (Fig. 2e). Longer protein half-lives and a greater absolute protein abundance compared to mRNA may account for the increased sensitivity of protein measurements. In addition, the protein assay may benefit from signal amplification because each antibody is conjugated with multiple DNA barcodes (an average of three DNA barcodes per antibody). There were shared differentially expressed proteins between this outlier cluster and the rest of the cells in all three donors (Fig. 2f), with a pattern resembling that of common myeloid progenitors (CMPs) (Fig. 2g), characterized by the high relative expression of CD34, CD38, CD123, CD117, CD13, CD33, and HLA-DR.
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Fig.2 REAP-seq characterization of ex vivo-activated naïve CD8+ T cells with aCD27. (Peterson, 2019)
Single Cell REAP-Seq Service
Creative Biolabs provides single cell REAP-seq service, using antibody labeling and droplet microfluidic systems to simultaneously analyze the transcriptome and proteome of single cells at high throughput. For more information, please contact us.
References
- Choi, J.R.; et al. Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses. Cells. 2020, 9(5): 1130.
- Peterson, V.M.; et al. Multiplexed quantification of proteins and transcripts in single cells. Nature Methods. 2017, 35: 936-939.
