Protocol for Identification and Removal of Doublets with DoubletDecon

Retention of multiplet captures in single-cell RNA sequencing (scRNA-seq) data can hinder identification of discrete or transitional cell populations and associated marker genes. To overcome this challenge, we created DoubletDecon to identify and remove doublets, multiplets of two cells, by using a combination of deconvolution to identify putative doublets and analyses of unique gene expression. Here, we provide the protocol for running DoubletDecon on scRNA-seq data. For complete details on the use and execution of this protocol, please refer to DePasquale et al. (2019). [Display omitted] •This protocol describes how to run and interpret the results of DoubletDecon•DoubletDecon uses deconvolution to identify and remove doublets in scRNA-seq data•Special focus on parameter tuning, data preparation, and troubleshooting•Optional steps for combining DoubletDecon results with alternative methods Retention of multiplet captures in single-cell RNA sequencing (scRNA-seq) data can hinder identification of discrete or transitional cell populations and associated marker genes. To overcome this challenge, we created DoubletDecon to identify and remove doublets, multiplets of two cells, by using a combination of deconvolution to identify putative doublets and analyses of unique gene expression. Here, we provide the protocol for running DoubletDecon on scRNA-seq data.