Proximity sequencing for the detection of mRNA, extracellular proteins and extracellular protein complexes in single cells

Complex cellular functions occur via the coordinated formation and dissociation of protein complexes. Functions such as the response to a signaling ligand can incorporate dozens of proteins and hundreds of complexes. Until recently, it has been difficult to measure multiple protein complexes at the single-cell level. Here, we present a step-by-step procedure for proximity sequencing, which enables the simultaneous measurement of proteins, mRNA and hundreds of protein complexes located on the outer membrane of cells. We guide the user through probe creation, sample preparation, staining, sequencing and computational quantification of protein complexes. This protocol empowers researchers to study, for example, the interplay between transcriptional states and cellular functions by coupling measurements of transcription to measurements of linked effector molecules, yet could be generalizable to other paired events. The protocol requires roughly 16 h spread over several days to complete by users with expertise in basic molecular biology and single-cell sequencing. Prox-seq is suitable for the analysis of primary single cells. The approach adopts various existing single-cell sequencing methods to enable characterization of proteins in close proximity (50-70 nm) along with mRNAs.The throughput of hundreds of targets simultaneously enables identification of protein targets forming complexes and is complementary to approaches such as FRET that sense macromolecules in contact over shorter distances. Proximity sequencing uses a panel of antibodies to probe single cells for up to hundreds of targets simultaneously. The approach is capable of detecting targets that are located within 50-70 nm of each other and thus likely to form complexes.