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Efficient profiling of total RNA in single cells with STORM-seq [cell lines]

ABSTRACT: Efficient profiling of total RNA in single cells with STORM-seq [cell lines]

PROVIDER: PRJNA1259241 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	SRR33445230.fastq.gz	Fastqsanger.gz
	SRR33445231_1.fastq.gz	Fastqsanger.gz
	SRR33445231_2.fastq.gz	Fastqsanger.gz
	SRR33445232_1.fastq.gz	Fastqsanger.gz

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Efficient profiling of total RNA in single cells with STORM-seq

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Project description:Despite significant advances, current single-cell RNA sequencing (scRNA-seq) technologies often struggle with accurately detecting non-coding transcripts, achieving full-length RNA coverage, and/or resolving transcript-level complexity. Many are also difficult to implement or inaccessible without specialized liquid handlers, further limiting their utility. We present Single-cell TOtal RNA-seq Miniaturized (STORM-seq), a random-hexamer primed, ribo-reduced single-cell total RNA sequencing (sc-total-RNA-seq) protocol using standard laboratory equipment. Adapted as a kit, STORM-seq constructs sequence-ready libraries in one working day, producing the highest complexity scRNA-seq libraries to-date, robustly measuring transcript isoforms and clinically relevant gene fusions in single cells. STORM-seq faithfully reconstructs expression profiles of locus-level transposable elements (TEs), and provides high-resolution profiling of transient, low-abundance enhancer RNAs (eRNAs), offering a powerful tool to dissect single-cell gene regulatory networks in unprecedented detail. Applied to human fallopian tube epithelium, the improved transcriptional resolution reveals a putative progenitor-like population and intermediate cell states, shaped by TEs and non-coding RNAs.

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Project description:Here we introduce STORM-seq, a standardized and generally available approach to profiling total RNA in single cells by miniaturization of the Takara SMART-seq Stranded (SSS) kit. The use of nanoliter-scale reaction volumes allows STORM-seq to efficiently scale to hundreds of high-complexity single cell libraries per run, from dissociated tissues and non-adherent cells, with greater than 99% per cell/library success. The use of ribosomal RNA depletion and random hexamer priming, rather than oligo-dT (poly-A) selection, allows comprehensive, whole-transcriptome analysis in each individual cell. STORM-seq recovers both coding and non-coding transcripts at an equal or greater rate to full volume reactions. Applying the method to cultured K562 cells and primary human fallopian tube epithelial (FTE) cells results in equivalent or better gene detection than manual preparation. Across all transcript biotypes, additional mRNA and lncRNA transcripts are detected beyond SSS or SMART-seq2. STORM-seq libraries from primary human FTE reveal intermediate cell types not observed by other technologies, with clear transitional significance visible upon data integration. STORM-seq provides a scalable, efficient protocol to recover deep whole transcriptomes from individual cells while minimizing costs.

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