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2.
SLAS Technol ; 27(2): 135-142, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35058211

RESUMEN

Next-generation sequencing (NGS) has revolutionized genomics, decreasing sequencing costs and allowing researchers to draw correlations between diseases and DNA or RNA changes. Technical advances have enabled the analysis of RNA expression changes between single cells within a heterogeneous population, known as single-cell RNA-seq (scRNA-seq). Despite resolving transcriptomes of cellular subpopulations, scRNA-seq has not replaced RNA-seq, due to higher costs and longer hands-on time. Here, we developed an automated workflow to increase throughput (up to 48 reactions) and to reduce by 75% the hands-on time of scRNA-seq library preparation, using the 10X Genomics Single Cell 3' kit. After gel bead-in-emulsion (GEM) generation on the 10X Genomics Chromium Controller, cDNA amplification was performed, and the product was normalized and subjected to either the manual, standard library preparation method or a fully automated, walk-away method using a Biomek i7 Hybrid liquid handler. Control metrics showed that both quantity and quality of the single-cell gene expression libraries generated were equivalent in size and yield. Key scRNA-seq downstream quality metrics, such as unique molecular identifiers count, mitochondrial RNA content, and cell and gene counts, further showed high correlations between automated and manual workflows. Using the UMAP dimensionality reduction technique to visualize all cells, we were able to further correlate the results observed between the manual and automated methods (R=0.971). The method developed here allows for the fast, error-free, and reproducible multiplex generation of high-quality single-cell gene expression libraries.


Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Automatización , ARN/genética , RNA-Seq , Análisis de la Célula Individual/métodos
3.
SLAS Discov ; 27(2): 140-147, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35093290

RESUMEN

Over the last decade, whole transcriptome profiling, also known as RNA-sequencing (RNA-seq), has quickly gained traction as a reliable method for unbiased assessment of gene expression. Integration of RNA-seq expression data into other omics datasets (e.g., proteomics, metabolomics, or epigenetics) solidifies our understanding of cell-specific regulatory patterns, yielding pathways to investigate the key rules of gene regulation. A limitation to efficient, at-scale utilization of RNA-seq is the time-demanding library preparation workflows, which is a 2-day or longer endeavor per cohort/sample size. To tackle this bottleneck, we designed an automated workflow that increases throughput capacity, while minimizing human error to enhance reproducibility. To this end, we converted the manual protocol of the NEBNext Directional Ultra II RNA Library Prep Kit for Illumina on the Beckman Coulter liquid handler, Biomek i7 Hybrid workstation. A total of 84 RNA samples were isolated from two human cell lines and subjected to comparative manual and automated library preparation methods. Qualitative and quantitative results indicated a high degree of similarity between libraries generated manually or through automation. Yet, there was a significant reduction in both hands-on and assay time from a 2-day manual to a 9-hour automated workflow. Using linear regression analysis, we found the Pearson correlation coefficient between libraries generated manually or by automation to be almost identical to a sample being sequenced twice (R²= 0.985 vs 0.983). This demonstrates that high-throughput automated workflows can be of great benefit to genomic laboratories by enhancing efficiency of library preparation, reducing hands-on time and increasing throughput potential.


Asunto(s)
ARN , Automatización , Biblioteca de Genes , Humanos , ARN Mensajero/genética , Reproducibilidad de los Resultados
4.
EMBO J ; 22(11): 2717-28, 2003 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-12773387

RESUMEN

Apoptosis is a hallmark event observed upon infection with many viral pathogens, including influenza A virus. The apoptotic process is executed by a proteolytic system consisting of a family of cysteinyl proteases, termed caspases. Since the consequences of apoptosis induction and caspase activation for the outcome of an influenza virus infection are not clear, we have addressed this issue by interfering with expression or function of a major virus-induced apoptosis effector, caspase 3. Surprisingly, influenza virus propagation was strongly impaired in the presence of an inhibitor that blocks caspase 3 and in cells where caspase 3 was partially knocked down by small interfering RNAs. Consistent with these findings, poor replication efficiencies of influenza A viruses in cells deficient for caspase 3 could be boosted 30-fold by ectopic expression of the protein. Mechanistically, the block in virus propagation appeared to be due to retention of the viral RNP complexes in the nucleus, preventing formation of progeny virus particles. Our findings indicate that caspase 3 activation during the onset of apoptosis is a crucial event for efficient influenza virus propagation.


Asunto(s)
Caspasas/metabolismo , Virus de la Influenza A/fisiología , Replicación Viral/fisiología , Animales , Secuencia de Bases , Butadienos/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/genética , Línea Celular , Chlorocebus aethiops , Inhibidores de Cisteína Proteinasa/farmacología , Perros , Activación Enzimática , Humanos , Virus de la Influenza A/patogenicidad , Nitrilos/farmacología , Oligopéptidos/farmacología , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacología , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Células Vero , Proteína Inhibidora de la Apoptosis Ligada a X
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