Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

We developed a tunable ligation technology to alter beads used for to capture mRNA for single-cell RNA sequencing (e.g. Drop-seq); we call this technique droplet assisted RNA targeting by single cell sequencing, or DART-seq. In the study, we applied the new technology to capture viral genome transcripts of recombinant type 3 Dearing orthoreovirus infecting murine L929 cells. In addition to capture of targeted viral gene transcripts, we were also able to simultaneosly pull-down polyadenylated mRNA from the L929 cells to observe host-pathogen interactions. We also applied the technology to efficiently measure natively paired heavy and light chain amplicons from B lymphocyte cells in a collection of human peripheral blood mononuclear cells. Overall design: Commercial Drop-seq beads (Chemgene) were altered by a tunable ligation chemistry to capture targets of interest while maintaining the ability to capture polyadenylated mRNAs. Altered or normal beads were resuspended in lysis buffer and were coencapsulated with single cells (either infected murine L929 cells or, in another experiment, human PBMCs) using a microfluidic device (as per standard Drop-seq protocol ; Macasko et al., 2015). cDNA was created from the captured RNA targets and amplified. Nextera library preparation was used to tagment and index the cDNA for next generation sequencing.