Immunity
Volume 53, Issue 4, 13 October 2020, Pages 878-894.e7
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Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

https://doi.org/10.1016/j.immuni.2020.09.015Get rights and content
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Highlights

  • Seq-Well S3 uses second-strand synthesis to improve transcript capture

  • Seq-Well S3 was benchmarked against a best-in-class commercial platform

  • Seq-Well S3 was applied to profile inflammatory cell states in skin diseases

  • Analysis of skin inflammation uncovered unique and conserved cellular phenotypes

Summary

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Keywords

single-cell RNA sequencing
scRNA-seq
Seq-Well
skin inflammation
acne
alopecia areata
granuloma annulare
leprosy
psoriasis

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