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Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

Hefei Zhang, Xuhang Li, Dongyuan Song, Onur Yukselen, Shivani Nanda, Alper Kucukural, Jingyi Jessica Li, Manuel Garber () and Albertha J. M. Walhout ()
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Hefei Zhang: University of Massachusetts Chan Medical School
Xuhang Li: University of Massachusetts Chan Medical School
Dongyuan Song: University of California
Onur Yukselen: Via Scientific Inc.
Shivani Nanda: University of Massachusetts Chan Medical School
Alper Kucukural: Via Scientific Inc.
Jingyi Jessica Li: University of California
Manuel Garber: University of Massachusetts Chan Medical School
Albertha J. M. Walhout: University of Massachusetts Chan Medical School

Nature Communications, 2025, vol. 16, issue 1, 1-21

Abstract: Abstract Transcriptomes provide highly informative molecular phenotypes that, combined with gene perturbation, can connect genotype to phenotype. An ultimate goal is to perturb every gene and measure transcriptome changes, however, this is challenging, especially in whole animals. Here, we present ‘Worm Perturb-Seq (WPS)’, a method that provides high-resolution RNA-sequencing profiles for hundreds of replicate perturbations at a time in living animals. WPS introduces multiple experimental advances combining strengths of Caenhorhabditis elegans genetics and multiplexed RNA-sequencing with a novel analytical framework, EmpirDE. EmpirDE leverages the unique power of large transcriptomic datasets and improves statistical rigor by using gene-specific empirical null distributions to identify DEGs. We apply WPS to 103 nuclear hormone receptors (NHRs) and find a striking ‘pairwise modularity’ in which pairs of NHRs regulate shared target genes. We envision the advances of WPS to be useful not only for C. elegans, but broadly for other models, including human cells.

Date: 2025
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DOI: 10.1038/s41467-025-60154-0

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