A SINC-Seq Protocol for the Analysis of Subcellular Gene Expression in Single Cells.

Microfluidic devices offer precise control of single cells and molecules by liquid flows, downsizing tools to allow us to perform single-cell assays at unprecedented resolutions and minimizing contamination. In this chapter, we introduce an approach, called single-cell integrated nuclear and cytoplasmic RNA-sequencing (SINC-seq), which enables precise fractionation of cytoplasmic and nuclear RNA of single cells. This approach uses electric field control in microfluidics to manipulate single cells and RNA sequencing to dissect gene expression and RNA localization in subcellular compartments. The microfluidic system for SINC-seq exploits a hydrodynamic trap (a constriction in a microchannel) to isolate a single cell, selectively lyses its plasma membrane via a focused electric field, and retains the nucleus at the hydrodynamic trap during the electrophoretic extraction of cytoplasmic RNA. Here, we provide a step-by-step protocol from microfluidic RNA fractionation to off-chip preparation of RNA-sequencing libraries for full-length cDNA sequencing using both a short-read sequencer (Illumina) and a long-read sequencer (Oxford Nanopore Technologies).

NanoSINC-seq dissects the isoform diversity in subcellular compartments of single cells.

Alternative mRNA isoforms play a key role in generating diverse protein isoforms. To dissect isoform usage in the subcellular compartments of single cells, we introduced an novel approach, nanopore sequencing coupled with single-cell integrated nuclear and cytoplasmic RNA sequencing, that couples microfluidic fractionation, which separates cytoplasmic RNA from nuclear RNA, with full-length complementary DNA (cDNA) sequencing using a nanopore sequencer. Leveraging full-length cDNA reads, we found that the nuclear transcripts are notably more diverse than cytoplasmic transcripts. Our findings also indicated that transcriptional noise emanating from the nucleus is regulated across the nuclear membrane and then either attenuated or amplified in the cytoplasm depending on the function involved. Overall, our results provide the landscape that shows how the transcriptional noise arising from the nucleus propagates to the cytoplasm.

Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells.

The physical fractionation of cytoplasmic versus nuclear components of cells is a key step for studying the subcellular localization of molecules. The application of an electric field is an emerging method for subcellular fractionation of proteins and nucleic acids from single cells. However, the multibiophysical process that involves electrical lysis of cytoplasmic membranes, electrophoresis, and diffusion of charged molecules remains unclear. Here we study RNA dynamics in single cells during the electrophoretic extraction via a microfluidic system that enables stringent fractionation of the subcellular components leveraging a focused electric field. We identified two distinct kinetics in the extraction of RNA molecules, which were respectively associated with soluble RNA and mitochondrial RNA. We show that the extraction kinetics of soluble RNA is dominated by electrophoresis over diffusion and has a time constant of 0.15 s. Interestingly, the extraction of mitochondrial RNA showed unexpected heterogeneity in the extraction with slower kinetics (3.8 s), while reproducibly resulting in the extraction of 98.9% ± 2% after 40 s. Together, we uncover that the microfluidic system uniquely offers length bias-free fractionation of RNA molecules for quantitative analysis of correlations among subcellular compartments by exploiting the homogeneous electrophoretic properties of RNA.

Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate).

We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.

SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology.

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.