rRNA depletion for holobiont metatranscriptome profiling across demosponges

The pervasive association of demosponges with diverse bacterial communities has been a research topic since the late 1970s. Studies characterizing these bacterial communities have detected over 40 bacterial phyla associated with sponges and determined that sponge microbiomes differ from the bacterioplankton of the surrounding water and include sponges-specific bacterial associates. Despite the large body of information available on the diversity of the sponge microbiome, the molecular mechanisms used by sponges to interact with their bacterial symbionts and vice versa remain poorly understood. Yet, studying how sponges interact with their microbiomes is pivotal to understanding how these systems react to environmental changes and when these changes lead to a disruption of sponge-microbe associations. An important factor hampering the characterization of the sponge-microbiome molecular cross-talk mechanisms is the lack of broadly applicable molecular methods to, for instance, efficiently sequence meta-transcriptomes across a broad range of sponge species. To overcome this limitation, we implemented a hybrid-capture strategy capable of depleting both sponge and bacterial rRNA from total RNA extracts of highly divergent demosponges with microbiomes of different complexity. Our pan-demosponge rRNA depletion strategy allows for the efficient characterization of the metatranscriptome of diverse demosponge holobionts and the simultaneous quantification of gene expression in both the host and its microbiome. ### Competing Interest Statement The pan-demosponge bait set was designed by siTOOLs in collaboration with the authors. The authors did not receive any payments or will profit from future siTOOLs sales. siTOOLs was not involved in the analysis of the data generated or in writing the manuscript, but provided information and feedback used for the methods section.