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

Area of Science:

• Genomics
• Molecular Biology
• Single-cell analysis

Background:

• Pooled genome editing with single-cell RNA sequencing (Perturb-seq) is powerful for genome function studies.
• Whole-transcriptome sequencing in Perturb-seq demands high depth, increasing costs and limiting accessibility.
• Existing methods face sensitivity and cost barriers for large-scale functional genomics screens.

Purpose of the Study:

• To introduce Targeted Perturb-seq (TAP-seq), an updated protocol addressing Perturb-seq's sensitivity and cost limitations.
• To provide a detailed protocol for performing cost-effective and scalable single-cell perturbation screens.
• To enable sensitive quantification of hundreds of specific transcripts instead of the whole transcriptome.

Main Methods:

• The TAP-seq workflow involves gene selection for targeted readout and primer design.
• Protocol details library preparation, pilot experiments, TAP-seq screening, and data analysis.
• Guidance is provided for designing targeted readout strategies and adapting TAP-seq to various single-cell RNA-sequencing platforms.

Main Results:

• TAP-seq yields single-cell measurements of selected gene and guide RNA expression levels.
• The method enables accurate guide RNA assignment to individual cells.
• Differential expression analysis reveals perturbation effects on target genes.

Conclusions:

• TAP-seq provides a sensitive, scalable, and cost-effective approach for single-cell perturbation screens.
• The protocol is adaptable to existing single-cell RNA-sequencing platforms.
• This method significantly lowers the barrier to entry for functional genomics research.