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ScBiopsy-seq: a platform for temporal single-cell RNA-seq analysis

ABSTRACT: The development of temporal single-cell RNA-seq (scRNA-seq) assay enabled us to systemically investigate the effects of various types of perturbations in a time course with single-cell resolution. However, the existing temporal scRNA-seq technologies have certain limitations in reliability, detection efficacy and detection diversity. In the current study, we develop scBiopsy-seq assay that combines a synergistic electroosmosis-electrophoresis extraction method for efficient RNA extraction and digital microfluidics with contamination-isolated hydrophobic interface for high-performance sample processing. scBiopsy-seq extracts the cytoplasm at well-controlled volume to detect >10K genes per extraction with 90% successful rate. Functional enrichment revealed that the genes robustly detected by scBiopsy-seq were associated to diverse biological processes, demonstrating its superb diversity of detection. scBiopsy-seq can perform the sequential extraction of the cytoplasm from the same cell multiple times, which allows us to associate the cell phenotypic responses with its transcriptic dynamics. We employed scBiopsy-seq to analyze the temporal response to BRD4 inhibition with single-cell resolution of SW480 cells and identified the key roles of fatty acid oxidation in this biological process. scBiopsy-seq with comparable data quality with scRNA-seq will dramatically expand the application of temporal scRNA-seq towards a broader spectrum of biological research.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE283737 | GEO | 2024/12/11

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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ScBiopsy-seq: a platform for temporal single-cell RNA-seq analysis

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