Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

  • Biology
  • Cell Atlas
  • Chemistry
  • Genomics
  • Immunology
  • R&D
  • Technology
  • Travis Hughes
  • Marc Wadsworth II
  • José Ordovas-Montañes
  • Alex K. Shalek
  • Hughes et al.▾
    Hughes, T.K., Wadsworth II, M.H., Gierahn, T.M., Do, T., Weiss, D., Andrade, P.R., Ma, F., de Andrade Silva, B.J., Shao, S., Tsoi, L.C., Ordovas-Montanes, J., Gudjonsson, J.E., Modlin, R.L., Love, J.C., Shalek, A.K.
  • Immunity , Volume 53 , Issue 4
  • October, 2020
Biology
Cell Atlas
Chemistry
Genomics
Immunology
R&D
Technology
Travis Hughes
Marc Wadsworth II
José Ordovas-Montañes
Alex K. Shalek

Abstract

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 Sincreased 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.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies