Alternative mRNA isoforms play a key role in generating diverse protein isoforms. To dissect isoform usage in the subcellular compartments of single cells, Researchers at the RIKEN Institute 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. These 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, these results provide the landscape that shows how the transcriptional noise arising from the nucleus propagates to the cytoplasm.
Workflow of NanoSINC-seq. (A) Single-cell isolation at a hydrodynamic trap via pressure-driven flow (t < 0 s). (B) Lysis of cell membrane and cytoplasmic RNA extraction via electrophoretic nucleic acid extraction (t = 0 s). (C) Sample extraction and library preparation for sequencing with long (ONT) and short (Illumina) read sequencers. Leading electrolyte (LE) and trailing electrolyte (TE) are aqueous buffers for the electrophoretic extraction with isotachophoresis (ITP) (12, 13, 32). P and E represent the pressure and electric field, respectively.