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LAST-seq:single-cell RNA sequencing by direct amplification of single-stranded RNA without prior reverse transcription and second-strand synthesis

ABSTRACT: Existing single-cell RNA sequencing (scRNA-seq) methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert single-stranded RNA into double-stranded DNA prior to amplification, with the limited RT/SSS efficiency compromising RNA detectability. Here, we develop a new scRNA-seq method, Linearly Amplified Single-stranded-RNA-derived Transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA molecules without prior RT/SSS. LAST-seq offers a high single-molecule capture efficiency and a low level of technical noise for single-cell transcriptome analyses. Using LAST-seq, we characterize transcriptional bursting kinetics in human cells, revealing a role of topologically associating domains in transcription regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE211836 | GEO | 2023/07/20

REPOSITORIES: GEO

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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

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LAST-seq:single-cell RNA sequencing by direct amplification of single-stranded RNA without prior reverse transcription and second-strand synthesis

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