Summary Metatranscriptomics is a powerful technique to study the ongoing processes in complex communities through the analysis of their gene expression profiles. As such, it is possible to assess, for example, whether there are inter- and intraspecific differences between the functioning of the microbiota of various host organisms, or how the functioning of the microbiome/microbiota may be altered under different conditions. This method has been used mostly to study free-living microbial communities (e.g. in sediment or water) and biofilms, as well as the microbiota of model organisms. However, metatranscriptomics approaches on non-model organisms from the marine environment are still relatively uncommon. Metatranscriptomic analysis of host tissue-associated microbial communities is namely complex as the amount of RNA originating from host cells generally greatly outnumbers the RNA originating from microbes. To avoid that the metatranscriptome sequencing data consists overwhelmingly of reads from the host rather than the microbiota and that very deep sequencing is required to offset this, it is important to remove as much host RNA (both coding polyA(+) messenger RNA and non-coding ribosomal RNA) from samples prior to sequencing library preparation. This is an additional step to the subtraction of ribosomal RNAs from microbes. As commercial kits for rRNA subtraction are not available for non-model organisms and their associated communities of prokaryotic and eukaryotic microbes, it is important to use tailormade sample-specific subtraction protocols. We studied the metatranscriptome of three octocoral species (Corallium rubrum, Eunicella cavolini, Paramuricea clavata) with a well-characterized microbiota composition. This required the development of a protocol to reduce rRNA and polyA(+)-mRNA from these host organisms. The protocol described here is a modification of the protocol used by Daniels et al. (2015), studying the metatranscriptome of a scleractinian coral, and is based on the protocol developed by Stewart et al. (2010). The protocol for the enrichment of bacterial mRNA for metatranscriptomics through rRNA and polyA(+)-mRNA subtraction consists of four stages: (1) the simultaneous extraction of both DNA and RNA from the same sample; (2) the development of biotin-labelled, sample-specific anti-sense rRNA probes targeting eukaryotic and prokaryotic rRNA using in vitro transcription; (3) the depletion of polyA(+)-mRNA from the total RNA sample using oligo(dT)25-coated beads to remove eukaryotic mRNA; followed by (4) subtractive hybridization for the removal of rRNA using the biotin-labelled anti-sense RNA probes. The RNA remaining after the polyA(+)-mRNA depletion and rRNA subtraction, will be relatively enriched in prokaryotic mRNA and is ready to use for sequencing library preparation.

The development of this protocol was supported through KAUST baseline funding to CRV, and through Chanel and the Fondation Paul Hamel to JvdW and CFP.