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bioRxiv · 10.64898/2026.08.25.747146

Decoding the Transcriptome Dark Matter: Construction of Single-Cell Whole-Transcriptome Regulatory Atlas by dropTotal

Liu, X.·Cao, W.·Pan, Y.·Luo, Z.·Wu, T.·Du, Y.·Xu, X.·Jin, Z.·Li, C.·Mu, Y.·Liu, Y.·Zhu, Q.
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Abstract

To profile unknown ncRNAs-"dark matter" in single cells, we developed dropTotal, a high-throughput droplet-based total RNA-seq method that uses dU-modified GAT primer with temperature-ramp hybridization and droplet merge barcoding to co-detect coding and non-coding transcripts with record sensitivity (>13,500 genes/cell, including >2,000 lncRNAs and >500 sncRNAs), compatible with fresh, frozen, fixed, and FFPE tissues. Applied to ~75,000 human glioma nuclei, it captured 60,313 genes (18,681 lncRNA, 19,859 mRNAs and 6,753 sncRNAs), enabling ncRNA-driven regulatory landscape construction. In oligodendroglioma, module analysis identified recurrence-associated ncRNA-centered modules linked to therapy resistance and invasion; in glioblastoma, six cellular states showed hundreds of state-specific unannotated ncRNAs with divergent functions, from MIR222HG-mediated immune modulation to SCIRT-driven hypoxia adaptation. Alternative splicing analysis identified 428 state-specific junction markers and mapped cell-state-specific alternative splicing regulation. dropTotal offers broad application for decoding the underlying ncRNA biology and single-cell whole transcriptome regulatory mechanisms in cellular identity and disease progression.

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Liu, X., Cao, W., Pan, Y., Luo, Z., Wu, T., Du, Y., Xu, X., Jin, Z., Li, C., Mu, Y., Liu, Y., Zhu, Q.. 2026-08-29. Decoding the Transcriptome Dark Matter: Construction of Single-Cell Whole-Transcriptome Regulatory Atlas by dropTotal. https://doi.org/10.64898/2026.08.25.747146

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