Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells

Abstract RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk...

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Autores principales:	Shreya Ghimire, Carley G. Stewart, Andrew L. Thurman, Alejandro A. Pezzulo
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c6a411eebcfd44aaae63f495fea96183
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spelling	oai:doaj.org-article:c6a411eebcfd44aaae63f495fea961832021-12-02T17:17:39ZPerformance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells10.1038/s41598-021-98912-x2045-2322https://doaj.org/article/c6a411eebcfd44aaae63f495fea961832021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-98912-xhttps://doaj.org/toc/2045-2322Abstract RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.Shreya GhimireCarley G. StewartAndrew L. ThurmanAlejandro A. PezzuloNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Shreya Ghimire Carley G. Stewart Andrew L. Thurman Alejandro A. Pezzulo Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
description	Abstract RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.
format	article
author	Shreya Ghimire Carley G. Stewart Andrew L. Thurman Alejandro A. Pezzulo
author_facet	Shreya Ghimire Carley G. Stewart Andrew L. Thurman Alejandro A. Pezzulo
author_sort	Shreya Ghimire
title	Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
title_short	Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
title_full	Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
title_fullStr	Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
title_full_unstemmed	Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells
title_sort	performance of a scalable rna extraction-free transcriptome profiling method for adherent cultured human cells
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c6a411eebcfd44aaae63f495fea96183
work_keys_str_mv	AT shreyaghimire performanceofascalablernaextractionfreetranscriptomeprofilingmethodforadherentculturedhumancells AT carleygstewart performanceofascalablernaextractionfreetranscriptomeprofilingmethodforadherentculturedhumancells AT andrewlthurman performanceofascalablernaextractionfreetranscriptomeprofilingmethodforadherentculturedhumancells AT alejandroapezzulo performanceofascalablernaextractionfreetranscriptomeprofilingmethodforadherentculturedhumancells
_version_	1718381168655597568

Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells

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