Abstract
Pooled genome editing combined with single-cell RNA sequencing—commonly known as Perturb-seq—has transformed the ability to interrogate genome function. However, whole-transcriptome single-cell RNA sequencing requires high sequencing depth to achieve the sensitivity needed for functional genomics screens, limiting its widespread use owing to prohibitive cost. Here we describe a detailed and updated protocol for targeted Perturb-seq (TAP-seq), a method that addresses the sensitivity and cost limitations of Perturb-seq. Instead of capturing the whole transcriptome, TAP-seq focuses on quantifying hundreds of transcripts of interest. The TAP-seq workflow involves first selecting genes for targeted readout, designing primers, conducting an initial pilot experiment and finally performing the TAP-seq screen and analyzing the data. We provide comprehensive guidance on designing targeted readout strategies for TAP-seq and describe all steps of the protocol, starting with library preparation. The outcome of TAP-seq is single-cell measurements of selected gene and guide RNA expression levels, guide RNA assignments to individual cells and differential expression results revealing perturbation effects on target genes. We further include instructions for adapting TAP-seq to all currently available single-cell RNA-sequencing platforms. Prior experience in single-cell technologies is beneficial and the protocol described can be completed in 2 days (excluding data analysis). In summary, this protocol describes how to perform sensitive, scalable and cost-effective single-cell perturbation screens.
Key points
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TAP-seq detects the effects of CRISPR perturbations across hundreds of target genes.
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TAP-seq is advantageous when sensitivity is key (such as for perturbations with small effect sizes or lowly expressed genes of interest) and costs are limiting.
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This protocol outlines strategies for designing targeted single-cell RNA-sequencing experiments, generating TAP-seq libraries and performing data analysis.
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Acknowledgements
We thank J. Reeve Baumann, D. Lindenhofer, J. Bezney and L. Velten for feedback on the manuscript. L.M.S. was supported by grants from the National Human Genome Research Institute of the National Institutes of Health (nos. RO1HG011664 and UM1HG011972) and the Dieter Schwarz Foundation Endowed Professorship. A.R.G. was supported by grants from the National Institutes of Health (nos. R01HG011664 and UM1HG011972).
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L.M.S. is co-founder and shareholder of Sophia Genetics, LevitasBio and Recombia Biosciences. The other authors declare no competing interests.
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Nature Protocols thanks Xin Jin, who co-reviewed with Xinhe Zheng, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference
Schraivogel, D. et al. Nat. Methods 17, 629–635 (2020): https://doi.org/10.1038/s41592-020-0837-5
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Supplementary Table 1 (download XLSX )
Oligonucleotide sequences.
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Moonen, D.P.I., Schraivogel, D., Gschwind, A.R. et al. Targeted single-cell RNA and perturbation sequencing with TAP-seq. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01367-5
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DOI: https://doi.org/10.1038/s41596-026-01367-5
