Linking two worlds: RNA and protein at single cell resolution
High-throughput single cell analysis lets researchers see the functions of complex biological systems. Detecting key proteins at the cell surface together with gene expression adds an additional layer of information. Reading the transcriptome and the proteome together at the single cell level reveals how one relates to the other, and how both perform cellular roles and functions. Drug development, research into infectious diseases and oncology are just some of the applications this new knowledge empowers.
Transcriptome and proteome research in one workflow
To incorporate proteomics data with the existing single cell transcriptomics GEXSCOPE technology, protein information can be converted into a nucleic acid sequence that can be sequenced in parallel with the transcriptome (1). Commonly, antibody-oligonucleotide conjugates (Ab-oligo, Figure 1) are used: antibodies detect specific cell surface proteins while the oligonucleotides allow compatibility with next generation sequencing (NGS) systems.
Figure 1: Cell stained with antibodies conjugates. Ab-oligos detecting cell surface proteins (right) and Ab-oligo containing PCR handle, antibody barcode and poly (A) tail (left).
The conjugated oligonucleotide sequence consists of a unique short sequence, the antibody-specific barcode, which enables identification of the target protein. The oligonucleotide also contains a 3’ poly (A) sequence, which can be captured with poly (T) probes. In addition, a universal PCR handle is used for the generation of NGS libraries.
For the combined detection of whole transcriptome and cell surface proteins, cells are first incubated with Ab-oligos. Stained cells are then partitioned on the Singleron SCOPE-chip®. Barcode Beads carrying poly (T) probes are then added to the microwells on the SCOPE-chip. Thereafter, the cellular mRNA together with Ab-oligos are captured onto the Barcode Beads allowing for cell-specific barcoding of each mRNA and Ab-oligo. Parallel mRNA and Ab-oligo NGS libraries are then generated. The Ab-oligo NGS libraries are used to determine the identity and quantity of the targeted proteins from individual cells (Figure 2).
Figure 2. Combined single cell transcriptomics and proteomics workflow.
Single cell characterization of peripheral blood mononuclear cells with an antibody cocktail
Compatibility of GEXSCOPE Single Cell RNA Library Kit with surface protein detection through Ab-oligos was demonstrated using the BD AbSeq Immune Discovery Panel (IDP). The IDP antibody cocktail contains antibodies against 30 human immune markers. Together, the GEXSCOPE Single Cell RNA Library Kits and BD AbSeq IDP panel were used to detect single cell surface proteins and transcriptome of peripheral blood mononuclear cells (PBMCs) (2).
PBMCs were clustered and annotated using mRNA data or mRNA and protein data together. Relying on transcript abundance, it was possible to determine five major cell types with T cells and monocytes being the most dominant cell types. Eight different T cell subtypes have been identified. Using transcript and protein abundance together, a better resolution of cells was achieved not only between the major cell types but also within cell populations (Figure 3).
Figure 3. UMAP clustering of PBMCs. Cells were colored based on cell type. A) UMAP coordinates were plotted for all PBMCs based on mRNA data only. B) UMAP coordinates were plotted for all PBMCs based on mRNA and protein data.
The correlation between mRNA and the respective proteins can differ significantly. It might be challenging to reliably reflect levels for transcripts with low expression levels or fast mRNA turn-over. In these cases, evaluation of protein abundance can provide deeper insights into the cell identity and/or ongoing molecular processes.
Figure 4. Detection of surface proteins with low mRNA expression. UMAP projection and cell types are the same as in Figure 3B. Color code corresponds to expression of mRNA (upper row) or protein (lower row). Expression levels of CD4 (A) and CD8 (B) are shown.
CD4 transcript abundance is generally low in CD4+ T cells and monocytes. However, high levels of CD4 protein in CD4+ T cells and moderate levels in monocytes have been observed at a protein level (Figure 6A). Similarly, examination of CD8 abundance revealed its higher expression in CD8+ T cells compared to CD8 expression at the mRNA level (Figure 6B).
For more information check our AppNote “RNA and protein at single cell resolution”.
1. Stoeckius, M., et al. (2017). Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865–868. https://doi.org/10.1038/nmeth.4380
2. BD Biosciences, BD AbSeq Immune Discovery Panel Brochure, https://www.bdbiosciences.com/en-de/products/reagents/single-cell-multiomics/abseq/abseq-immune-discovery-panel
Acknowledgement: We thank BD for providing the antibodies and their advice for downstream data analysis.
Copyright © 2023 Singleron Biotechnologies