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

## TL;DR

This paper introduces a new microfluidic method called SplitBlot that allows simultaneous analysis of DNA and nuclear/cytoplasmic proteins from the same single cell.

## Contribution

SplitBlot enables paired multiomic measurements of genomic DNA and nucleo-cytoplasmic proteins from individual cells using microfluidic technology.

## Key findings

- SplitBlot successfully amplified TurboGFP DNA from 86% of agarose-encapsulated DNA pallets.
- Nuclear histone H3 and cytoplasmic beta-actin were resolved with a separation resolution of R_s = 0.77.
- The method was validated using U251 cells engineered to express TurboGFP.

## Abstract

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.