Preservation of full-length transcripts at ambient temperature using ensilication
Muraleedharan, M.; Gowda, H.; Banal, J.
Show abstract
Long-read RNA sequencing can resolve full-length transcript isoforms directly from single molecules, but this capability is contingent on maintaining molecular integrity. Here we show that ensilication, the encapsulation of RNA within a deposited silica matrix, preserves isoform-level fidelity in PacBio Kinnex long-read sequencing after seven days of storage at 40 {degrees}C. Pooled human total RNA was profiled across six preservation conditions spanning cryogenic freezing, ensilication at ambient and elevated temperatures, and unprotected storage under identical thermal stresses. Ensilicated RNA was indistinguishable from frozen controls across gene and isoform quantification, differential expression, dominant isoform identity, and splice junction phasing. Unprotected RNA under the same conditions lost half its detectable isoforms, showed dominant isoform switching in over half of multi-isoform genes, and lost all gene-spanning splice junction arcs at individual loci. This failure traces to directional 5 truncation, a mode of information loss specific to oligo-dT primed long-read workflows and invisible to the short-read methods used to validate prior preservation approaches. These results establish that isoform-resolution transcriptomics can be decoupled from cold-chain infrastructure.
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