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Nucleic Acids Res. 2019 Feb 20;47(3):e16. doi: 10.1093/nar/gky1173.

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

Author information

1
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
2
Section of Rheumatology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520, USA.
3
Department of Dermatology, Yale University, School of Medicine, New Haven, CT, USA.
4
Department of Pathology, Yale University, School of Medicine, New Haven, CT, USA.
5
Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA.
6
Yale Stem Cell Center, Yale Cancer Center, New Haven, CT 06520, USA.

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 challenging despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing single-cell freeze-thaw lysis directly toward 3' mRNA sequencing. 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 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 3' scRNA-seq. We applied it 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 library preparation, we envision it to allow sampling at the distributed sites including point-of-care settings and downstream processing at centralized facilities, which should enable wide-spread adoption beyond academic laboratories.

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