Abstract
Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host–pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.
Highlights
Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods
We have applied dual RNA-seq to human epithelial cells subjected to the bacteria Chlamydia trachomatis, which is an obligate intracellular, human-specific bacterial pathogen that causes trachoma and urogenital infections[8,9,10]
Dual RNA-seq was applied to C. trachomatis serovar E-infected human Human epithelial type 2 (HEp-2) epithelial cells in triplicate at 1 and 24 h post-infection
Summary
Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. It is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered This data highlights the delicate nature of balancing host–pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights. The previous chlamydialbased dual RNA-seq experiment encompassed an experimental design that used a multiplicity of infection (MOI) of 1, while their depletion technique removed rRNAs in all samples, followed by subjecting half of these libraries to polyA depletion to further enrich chlamydial transcripts. A higher MOI will likely affect the developmental cycle due to the underlying stress this places on host cells[25,26]
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