Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Abstract

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Single cell protein analysis for systems biology

“…Another approach for increasing the antibody readouts per cell relies on conjugating antibodies with DNA oligonucleotides (barcodes), and a number of recent methods, including Abseq, cellular indexing of transcriptome and epitope by sequencing (CITE-seq), and RNA expression and protein sequencing (REAP-seq), have employed this strategy [48,53,54] (see Figure 1 and Table 1). With Abseq, single cells are incubated with barcoded antibodies recognizing cell-surface proteins; the method is limited to surface proteins.…”

Single-cell technologies for profiling T cells to enable monitoring of immunotherapies

“…These two methods can be readily adapted to different high-throughput scRNA-seq platforms. Another similar technique that can be expanded to demonstrate the same capability is called Abseq (Figure 1D), which utilizes a combination of DNA-labeled antibody and droplet microfluidics [71]. One disadvantage of all three of these approaches is that the information about the spatial distribution of proteins is lost.…”

Bulk double emulsification for flow cytometric analysis of microfluidic droplets

“…Microfluidic droplet compartmentalization affords powerful strategies for biochemical assays, 1 sequencing large numbers of single cells, 2,3 generating quantitative libraries from low-input samples, 4 and creating uniform protein crystals for X-ray scattering. 5 Other applications rely on the ability to perform reactions in droplets, then analyze or sort them.…”

“…They use similar principles to measure and sort cells and beads, but are far superior in speed, multiplexing, and sensitivity. 1 However, they cannot operate on microfluidic emulsions carried in oil, since they are designed for biological samples, carried in water. This has inspired methods for re-dispersing water-in-oil droplets into water carriers, allowing FACS analysis.…”