Work

scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3′ mRNA profiling

Year: 2018

Type: article

Abstract: ABSTRACT Cellular barcoding of 3’ mRNAs enabled massively parallel profiling of single-cell gene expression and has been implemented in droplet and microwell based platforms. The latter further adds the value for compatibility with low input samples, optical imaging, scalability, and portability. However, cell lysis in microwells remains suboptimal despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing s ingle- c ell f reeze- t haw lysis d irectly toward 3’ mRNA seq uencing. It offers format flexibility with a simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and the cost per sample matching that of the state-of-the-art scRNA-seq platforms. Freeze-thaw, known as an unfavorable lysis method resulting in possible RNA fragmentation, turns out to be fully compatible with single-cell 3’ mRNA sequencing, which detects only ~50 bases at the 3’ end. We applied it to the profiling of mixed populations including whole tumors for distinguishing all major cell types and to the profiling of circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis. Our results delineate the heterogeneity in the transcriptional programs and effector functions of these rare pathogenic T cells. As scFTD-seq decouples on-chip cell isolation and the following library preparation steps, we envision it to potentially allow the sampling (capture of cells/beads in microwells) at the distributed sites including small clinics or point-of-care settings and downstream processing at a centralized facility, which should enable wide-spread adoption beyond academic laboratories – for any users even with no experience in scRNA-seq library generation.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gky1173