scPASU: A computational protocol for quantifying polyadenylation site usage and alternative polyadenylation from 3′ scRNA-seq data

3′ single-cell RNA sequencing (scRNA-seq) captures polyadenylation (poly(A)) sites, enabling quantification of site usage per gene and cell. Here, we present scPASU (single-cell poly(A) site usage), a Snakemake workflow for quantifying poly(A) site usage and alternative polyadenylation from 3′ scRNA-seq data. We describe steps for building a poly(A) site reference, generating a site-by-cell matrix per sample, and testing alternative polyadenylation (APA) between cell groups. This protocol is configurable for organism- and sample-specific parameters and supports discovery of poly(A) sites. For complete details on the use and execution of this protocol, please refer to Le et al. 1 • Workflow to generate a novel poly(A) isoform reference from scRNA-seq data • Steps for creating a cell-by-poly(A) peak matrix from Cell Ranger BAM files • Guidance on performing alternative polyadenylation testing between different cell types Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 3′ single-cell RNA sequencing (scRNA-seq) captures polyadenylation (poly(A)) sites, enabling quantification of site usage per gene and cell. Here, we present scPASU (single-cell poly(A) site usage), a Snakemake workflow for quantifying poly(A) site usage and alternative polyadenylation from 3′ scRNA-seq data. We describe steps for building a poly(A) site reference, generating a site-by-cell matrix per sample, and testing alternative polyadenylation (APA) between cell groups. This protocol is configurable for organism- and sample-specific parameters and supports discovery of poly(A) sites.