RNAseq of Infected Erythrocyte Surface Antigen-Encoding Genes.

Autor:	Nguyen HHT; Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC, Australia.; Peter Doherty Institute, Melbourne, VIC, Australia.; Bio21 Institute, Parkville, VIC, Australia., Azizan S; Bio21 Institute, Parkville, VIC, Australia.; School of BioSciences, The University of Melbourne, Parkville, VIC, Australia., Yeoh LM; Peter Doherty Institute, Melbourne, VIC, Australia.; Bio21 Institute, Parkville, VIC, Australia.; Department of Microbiology and Immunology, School of Biomedical Sciences, The University of Melbourne, Parkville, VIC, Australia., Tang J; School of Medicine, Faculty of Health, Deakin University, Waurn Ponds, VIC, Australia., Duffy MF; Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC, Australia. mduffy@unimelb.edu.au.; Peter Doherty Institute, Melbourne, VIC, Australia. mduffy@unimelb.edu.au.; Bio21 Institute, Parkville, VIC, Australia. mduffy@unimelb.edu.au.; Department of Microbiology and Immunology, School of Biomedical Sciences, The University of Melbourne, Parkville, VIC, Australia. mduffy@unimelb.edu.au.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2470, pp. 185-209.
DOI:	10.1007/978-1-0716-2189-9_15
Abstrakt:	Massive parallel sequencing technology has greatly increased the breadth and depth of transcriptomic data that can be captured from P. falciparum samples. This has revolutionized in vitro studies but uptake has been slower in the analysis of clinical samples. The principal barriers are the removal of contaminating white blood cells in a malaria endemic setting and preservation of the RNA. We provide here detailed methods for the collection of purified infected erythrocytes and the preservation and extraction of RNA. We also provide methods for assessing and addressing contaminating RNA from erythroid cells, and a protocol for RNAseq library preparation optimized to maximize yield from low amounts of parasite mRNA. Finally, we provide some examples of RNAseq library characteristics that may fail quality control for other species but are in fact satisfactory for P. falciparum RNAseq. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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