What question did this study set out to answer?

To develop a framework for total RNA sequencing that captures both coding and noncoding RNA across multiple cell types in the human brain and other tissues.

April 3, 2026Open Access

Scalable single-cell total RNA sequencing unifies coding and noncoding transcriptomics

AIAlina IsakovaStanford Medicine DLDaniel Dan LiuCalifornia Institute for Regenerative Medicine ICIvana CvijovićStanford University

Key Points

To develop a framework for total RNA sequencing that captures both coding and noncoding RNA across multiple cell types in the human brain and other tissues.
Developed a total RNA profiling framework for droplet-based sequencing platforms.
Applied the framework to analyze developing human brain tissues and peripheral blood mononuclear cells.
Investigated microRNA dynamics in specific neuron types and dengue-infected hepatocytes.
Mapped diverse RNA biotypes, revealing specific expression programs linked to cell types and development stages.
Showed enrichment of MIR137 in Cajal-Retzius and early-born neurons, suggesting potential links to schizophrenia.
Identified transcriptional modules that combine coding and noncoding RNAs in human blood cells, enhancing understanding of gene regulation.

Abstract

Current single-cell RNA atlases largely capture polyadenylated transcripts while missing critical regulatory layers from noncoding RNA. To address this, we develop a generalizable framework that adapts total RNA profiling for use in standard droplet-based platforms and captures a broad complement of coding and noncoding RNAs using a unified pipeline. Applying this approach to the developing human brain, we generate a dataset mapping diverse RNA biotypes across all neuronal and non-neuronal lineages, revealing biotype-specific expression programs with cell-type and temporal specificity. Tracking microRNA dynamics in Cajal-Retzius neurons, transient and early-born neurons in the cortex, we show the enrichment and target anticorrelation of MIR137, associated with schizophrenia and intellectual disability, suggesting tight regulatory control. We apply TotalX to human peripheral blood mononuclear cells and identify transcriptional modules combining coding and noncoding RNAs and tRNA dynamics. In addition, we analyze dengue-infected hepatocytes and capture non-adenylated viral transcripts that distinguish infection states. This expanded coverage helps with understanding cellular identity and gene regulation at the atlas scale.

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