Fig. 1

Parallel preparation of thousands of single cells by nPOP. a The slide-based approach allows programming different droplet layouts, such as the 4 examples shown in the picture. b Four slides with 2016 single cells from an nPOP experiment using droplet configuration AL-01. Samples are surrounded by a perimeter of water droplets for local humidity control. Slides are placed on a cooling surface to further prevent evaporation. c A schematic of the nPOP method illustrates the steps of cell lysis, protein digestion, peptide labeling with TMTpro, quenching of labeling reaction, and sample collection. These steps are performed for each single cell (corresponding to a single droplet). d After labeling, single-cell samples are automatically pooled into sets and transferred into a 384-well plate, which is then placed into the autosampler for automated injection for LC-MS/MS. Any system that supports 384-well plate injection (such as Dionex 3000) can implement this workflow