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), which 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 utilized 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 the 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, SplitBlot 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.