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Ghimire S, Stewart CG, Thurman AL, et al. Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells. Sci Rep. 2021;11(1):19438doi: 10.1038/s41598-021-98912-x.
Ghimire, S., Stewart, C. G., Thurman, A. L., & Pezzulo, A. A. (2021). Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells. Scientific reports, 11(1), 19438. https://doi.org/10.1038/s41598-021-98912-x
Ghimire, Shreya, et al. "Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells." Scientific reports vol. 11,1 (2021): 19438. doi: https://doi.org/10.1038/s41598-021-98912-x
Ghimire S, Stewart CG, Thurman AL, Pezzulo AA. Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells. Sci Rep. 2021 Sep 30;11(1):19438. doi: 10.1038/s41598-021-98912-x. PMID: 34593905; PMCID: PMC8484438.
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Sci Rep. 2021 Sep 30;11(1):19438. doi: 10.1038/s41598-021-98912-x.

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

Scientific reports

Shreya Ghimire, Carley G Stewart, Andrew L Thurman, Alejandro A Pezzulo

Affiliations

  1. Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
  2. Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA. [email protected].

PMID: 34593905 PMCID: PMC8484438 DOI: 10.1038/s41598-021-98912-x

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.

© 2021. The Author(s).

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