title: RNA-Seq of ARMS bulk sessile fraction
design: The standard ARMS processing protocol (Ransome et al., 2017) was modified in order to test our specific hypothesis. From each ARMS unit, five fractions were collected: the photosynthetic community, the holobiont community, the environmental (sediment) microbiome, the Halisarca sponge microbiome, and the Tethya sponge microbiome. To do this ARMS were removed from their holding tanks, and the 9 plates (17 plate surfaces as there is no accessible bottom surface to the bottom plate) were separated and rinsed to dislodge loosely attached organisms. The water and previously trapped sediment in the holding tank were retained.   First, the photosynthetic community was sampled by randomly subsampling (4 x 1 cm2) the top surface of the top ARMS plate (e.g., Figure 2C). Second, the two sponge fractions (Halisarca sp. and Tethya sp.; e.g., Figure 2E) were generated by sampling morphologically identified sponges from the internal plates. Both are low-microbial abundance sponges (Gloeckner et al., 2014; Lesser et al., 2020), and Tethya sp. was only found at Upa-Upasina. Thirdly, the holobiont community fraction was generated by scraping, and blending the scrapings from all 17 plates, including the remainder of the light-exposed top surface of the top plate and all surfaces of the remaining 8 shaded plates (e.g., Figure 2D). While this fraction will include both photosynthetic and non-photosynthetic organisms, the shaded plate surfaces constitute ~16x the area of the light-exposed top surface of the top plate. This homogenised bulk sample was then subsampled (50ml). Finally, the environmental fraction was generated by passing the water and sediment from the holding tank through a 500m and then a 100m sieve, and collecting the material which did not pass the 100m sieve, which was then subsampled (10g). This fraction was therefore primarily composed of sediment; microbes, including free-living sediment dwelling microbes (e.g., bacteria); and microplankton (e.g., single celled algae such as diatoms and dinoflagellates.   From each ARMS unit each fraction was split in two and one half was snap frozen for metabolomic analysis and the other was placed in RNA Later for metabarcoding. All samples were returned to the Smithsonian National Museum of Natural History (Washington, DC, USA) in a liquid nitrogen dry shipper. Total DNA was extracted from 10g of the holobiont community samples and 5g of the environmental samples using a MO-BIO Powermax Soil DNA Isolation Kit according to the manufacturers protocol with the addition of 400g/ml Proteinase K and an overnight lysis step at 56C and 200rpm. The photosynthetic  community and sponge subsamples were extracted with the DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturers instructions. All DNA extracts were purified using MO-BIO PowerClean DNA Clean-Up Kits, quantified (Qubit dsDNA HS Kit), run on an agarose gel, and DNA quality investigated using ImageJ software. All sample types are known to contain relatively high bacterial biomass and thus we do not expect contamination from the lab environment or equipment to be a major issue in DNA libraries. However, extraction and PCR amplification controls were included for all sample types, and were all negative and so were not sequenced. Each sample was analysed with 16s rRNA gene metabarcoding (for the microbe community) and mass spectrometry (for metabolomics). All 16S rRNA gene libraries were prepared for sequencing using the original Earth Microbiome protocol using primers 515F and 806 R (Caporaso et al., 2012). To investigate the photosynthetic community, 23S rRNA gene libraries were also prepared using the protocol described in Marcelino and Verbruggen (2016) using a two-step PCR procedure that first amplifies the gene fragment followed by ligation of the barcoded Illumina adaptors to the amplicons in a second PCR reaction.