DART-seq

Abstract Here we describe the step-by-step protocol of Droplet Assisted RNA Targeting by single cell sequencing \(DART-seq). DART-seq allows simultaneous multiplexed amplicon sequencing and transcriptome analysis in single cells. Using a simple and quick modification of commercially available barcoded beads we have expanded the application of droplet microfluidics based high throughput single cell RNA sequencing technology to include non-polyadenylated RNA transcripts. Specific probes targeting RNA of interest were attached to the beads using a T4 DNA Ligase based enzymatic reaction.The modified DART-seq beads were then used in standard Drop-seq platform to generate single cell libraries for sequencing. We have applied DART-seq to analyze reovirus RNA sequences in infected murine L cells. As a second application we used DART-seq to investigate the B cell repertoire in human peripheral blood mononuclear cell \(PBMC) samples.