Prox-seq: Simultaneous quantification of proteins, protein complexes and mRNA in single-cells via proximity-sequencing

A novel assay based on proximity ligation assay for high-throughput quantification of proteins, protein complexes and mRNA in single cells Here we present proximity-sequencing (Prox-seq), a method for simultaneous measurement of an individual cell’s proteins, protein complexes and mRNA. Prox-seq utilizes deep sequencing and barcoded proximity assays to measure proteins and their complexes from all pairwise combinations of targeted proteins, without any prior knowledge of which proteins can form complexes. The number of measured protein complexes scales quadratically with the number of targeted proteins, allowing for unparalleled multiplexing capacity. We developed a high-throughput experimental and computational pipeline and demonstrated the potential of Prox-Seq for multi-omic analysis with a panel of 13 proximity probes, enabling the measurement of 91 protein complexes, along with thousands of mRNA molecules in single T cells and B cells. Prox-seq is compatible with current single-cell RNA sequencing assays, such as Drop-seq and Smart-seq2. Prox-seq provides access to an untapped yet powerful measurement modality for single-cell phenotyping and has the potential to discover new protein interactions in single-cells. mRNA and PLA profiling of single Jurkat and Raji cells. PLA profiling of single human PBMCs. PLA profiling of single primary human macrophages after stimulation with LPS, PAM2CSK4, or both for 5 minutes, 2 hours or 12 hours. Prox-seq profiling of single human PBMCs using 10x pipeline