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Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a  Wolbachia-Drosophila  system, this bacterial mRNA enrichment yielded a 3-fold increase in  Wolbachia  mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from  Ehrlichia -infected canine cells covers 93% of the  Ehrlichia  genome, suggesting ubiquitous transcription across the entire  Ehrlichia chaffeensis  genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.

Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples