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<p>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.</p>

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Targeted single-cell RNA and perturbation sequencing with TAP-seq

  • 期刊论文

  • 发表日期：

    2026年5月26日

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  • Dewi P. I. Moonen,
  • Daniel Schraivogel,
  • Andreas R. Gschwind,
  • Lars M. Steinmetz

摘要

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.