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The article presents a study of the use of nanopore sequencing with single-cell integrated nuclear and cytoplasmic ribonucleic acid (RNA) sequencing (NanoSINC-seq) in dissecting isoform diversity in subcellular compartments of single cells. Topics discussed include the procedures used in preparing cytoplasmic RNA and nuclear RNA from the single K562 cells, role of nuclear export in the transcriptional abundance in the cytoplasm, and the throughput limitation of the NanoSINC-seq approach.

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)., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)