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Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies
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 acc...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cell Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562821/ https://www.ncbi.nlm.nih.gov/pubmed/33053333 http://dx.doi.org/10.1016/j.immuni.2020.09.015 |
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author | Hughes, Travis K. Wadsworth, Marc H. Gierahn, Todd M. Do, Tran Weiss, David Andrade, Priscila R. Ma, Feiyang de Andrade Silva, Bruno J. Shao, Shuai Tsoi, Lam C. Ordovas-Montanes, Jose Gudjonsson, Johann E. Modlin, Robert L. Love, J. Christopher Shalek, Alex K. |
author_facet | Hughes, Travis K. Wadsworth, Marc H. Gierahn, Todd M. Do, Tran Weiss, David Andrade, Priscila R. Ma, Feiyang de Andrade Silva, Bruno J. Shao, Shuai Tsoi, Lam C. Ordovas-Montanes, Jose Gudjonsson, Johann E. Modlin, Robert L. Love, J. Christopher Shalek, Alex K. |
author_sort | Hughes, Travis K. |
collection | PubMed |
description | 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 S(3) (“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 S(3) 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 S(3) to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation. |
format | Online Article Text |
id | pubmed-7562821 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Cell Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-75628212020-10-20 Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies Hughes, Travis K. Wadsworth, Marc H. Gierahn, Todd M. Do, Tran Weiss, David Andrade, Priscila R. Ma, Feiyang de Andrade Silva, Bruno J. Shao, Shuai Tsoi, Lam C. Ordovas-Montanes, Jose Gudjonsson, Johann E. Modlin, Robert L. Love, J. Christopher Shalek, Alex K. Immunity Resource 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 S(3) (“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 S(3) 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 S(3) to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation. Cell Press 2020-10-13 /pmc/articles/PMC7562821/ /pubmed/33053333 http://dx.doi.org/10.1016/j.immuni.2020.09.015 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Resource Hughes, Travis K. Wadsworth, Marc H. Gierahn, Todd M. Do, Tran Weiss, David Andrade, Priscila R. Ma, Feiyang de Andrade Silva, Bruno J. Shao, Shuai Tsoi, Lam C. Ordovas-Montanes, Jose Gudjonsson, Johann E. Modlin, Robert L. Love, J. Christopher Shalek, Alex K. Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title | Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title_full | Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title_fullStr | Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title_full_unstemmed | Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title_short | Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies |
title_sort | second-strand synthesis-based massively parallel scrna-seq reveals cellular states and molecular features of human inflammatory skin pathologies |
topic | Resource |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562821/ https://www.ncbi.nlm.nih.gov/pubmed/33053333 http://dx.doi.org/10.1016/j.immuni.2020.09.015 |
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