Paired Analyses of Nuclear Protein Targets and Genomic DNA by Single-Cell Western Blot and Single-Cell PCR

Single-cell multimodal assays measure multiple layers of molecular information. Existing single-cell tools have limited capability to analyze nuclear proteins and genomic DNA from the same originating single cell. To address this gap, we designed and developed a microfluidic single-cell assay (SplitBlot), that pairs measurements of genomic DNA (PCR-based) and nucleo-cytoplasmic proteins (nuclear histone H3 and cytoplasmic beta-actin). To accomplish this paired multiomic measurement, we utilize microfluidic precision to fractionate protein molecules (both nuclear and cytoplasmic) from genomic DNA (nuclear). We create a fractionation axis that prepends a comet-like encapsulation of genomic DNA in an agarose molded microwell to a downstream single-cell western blot in polyacrylamide gel (PAG). For single-cell genomic DNA analysis, the agarose-encapsulated DNA is physically extracted from the microfluidic device for in-tube PCR, after release of genomic DNA from a molten agarose pallet (86% of pallets resulted in amplification of TurboGFP). For protein analysis, nucleo-cytoplasmic proteins are photocaptured to the PAG (via benzophenone) and probed in-situ (15 kDa histone H3 resolved from 42 kDa beta-actin with a separation resolution R _s = 0.77, CV = 76%). The SplitBlot reported the amplification of TurboGFP DNA and the separation of nuclear histone H3 and cytoplasmic beta-actin from the same single U251 cells engineered to express TurboGFP. Demonstrated here, Split-Blot offers the capacity for precision genomic DNA vs. protein fractionation for subsequent split workflow consisting of in-tube PCR and on-chip single-cell western blotting, thus providing a tool for pairing genotype to nuclear and cytoplasmic protein expression at the single-cell level.