Evaluation of single‐cell sorting accuracy using antibody‐derived tag‐based qPCR

Abstract

AbstractSingle‐cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence‐activated cell sorting (FACS) for downstream applications such as single‐cell sequencing or single‐cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single‐cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co‐staining cells with the same marker using both antibody‐derived tags (ADTs) and fluorescent antibodies. After single‐cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single‐cell level. Finally, we applied this assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT‐based qPCR assay developed here can be utilized to confirm single‐cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate.