Detection of Nuclear RNA Decay Intermediates Using a Modified Oxford Nanopore RNA Sequencing Strategy

The nuclear RNA exosome complex is crucial for noncoding RNA processing and RNA quality control in the nucleus. Identifying substrates and intermediates of RNA decay pathways, such as those mediated by the exosome complex using Oxford Nanopore sequencing can be difficult in part because a simple method to detect them has been lacking and also because some of these RNAs lack abundant poly(A) tails which are required for Oxford Nanopore-based sequencing. Here we describe an Oxford nanopore-based approach which can be used to identify long reads corresponding to precursors and products of nuclear exosome processing. We are able to observe accumulation of unprocessed snoRNAs, cleavage products of the yeast nuclear RNase III endonuclease Rnt1p when the nuclear exosome component Rrp6p is inactivated.