Combining the analysis of protein biomarkers to genome-wide expression profiling at the single-cell level provides deeper scientific insights than previously possible. Despite being widely adopted, CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing), the technique that enables this combination, still needs standardization and simplification to help overcome some of its challenges such as the lack of quality checks to test the binding of antibodies to report expression of biomarkers before proceeding with the whole CITE-Seq assay.
 

Usually, single-cell multiomic protocols involve specifically isolating the cell population of interest with fluorescence activated cell sorting (FACS) and then using that population for simultaneous single-cell sequencing of gene (RNA-Seq) and protein expression (CITE-Seq). This requires co-staining of cells with antibodies conjugated to both fluorescent dyes for flow cytometry analysis and sorting and oligonucleotides for CITE-Seq. This means that, in addition to the already mentioned uncertain binding of oligo-conjugated antibodies, this procedure also poses the risk of antibody competition and eventual compromise of the reporting signal intensity.
 

Shi et al. addressed these issues in a recent publication and provided an easy-to-use workflow utilizing a select set of fluorescent dye-oligo conjugates complementary to the specific antibody-derived tags (ADTs) used to perform the CITE-Seq assay. The strategy was called a flow proxy assay since it allowed confirmation of ADT binding to cells using flow cytometry prior to the single-cell capture, library preparation and sequencing workflows. As a proof of principle, the authors applied this method to different experimental scenarios from a single ADT marker detection up to a 10-marker multiplex detection approach, evaluating multiple biomarkers simultaneously. The authors also demonstrated the applicability of this strategy to selectively sort natural killer (NK) and T cells using a 5-marker panel of dye-oligo conjugates and fluorescent activated cell sorting followed by CITE-Seq.