title: Messenger RNA Sequencing of Rare Cell Populations in the Lung and Lung-Draining Lymph Nodes.
abstract: Next-generation sequencing (NGS) techniques provide unique prospects for in-depth transcriptome analyses. Nevertheless, the emerging and still growing knowledge about the large diversity and heterogeneity of cells that participate in immunological responses in a tissue- and micromilieu-specific manner calls for advanced isolation and sequencing methods for the accurate quantification of gene expression in small cell populations and even individual cells from any organ or tissue. One of the major limitations in performing transcriptome analyses of rare cell populations was and still is quality and quantity of RNA that often limits analyses of complex mixtures of immune cell populations. Here, we describe a protocol to isolate rare T cell populations from the lung and in particular the subsequent methods to isolate high-grade RNA in order to perform NGS-based transcriptome analyses.
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ulges
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany.
authors.forename: Edgar
authors.initials: E
authors.lastname: Schmitt
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany. boppt@uni-mainz.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Bopp
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Klein
mesh_terms: D000818:Animals; D002469:Cell Separation; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D018740:Genetic Heterogeneity; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008168:Lung / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger
keywords: Lung; Rare T cell population; Transcriptome analysis