title: RNA-Seq of ARMS
design: On collection, a 106m nitex-lined crate was placed over each ARMS on the seafloor, and they were returned to the surface where each ARMS was placed in an individual holding tank with 45m filtered aerated seawater. ARMS were transported to the boat or shore where they were sampled rapidly to minimize molecular degradation. ARMS were removed from their holding tanks and plates were separated, rinsed to dislodge loosely attached organisms, which were retained along with the water and sediment in the holding tank. The sessile communities from the plates were processed by scraping, blending, subsampling (50ml) and storing at -80C. Motile communities were generated by removing organisms larger than 2mm, and then passing the water and sediment from the holding tank through first a 500m sieve and secondly a 100m sieve, and collecting the material which did not pass through each sieve.  This produced samples in three fractions: a sessile fraction; a 100m-500m motile fraction; and a 500m-2mm motile fraction. Sessile fractions were placed in DMSO and motile fractions were placed in EtOH for metabarcoding. All samples were returned to the Smithsonian National Museum of Natural History (Washington, DC, USA) and stored at -80C until they were processed.  Total DNA was extracted from 10g of the sessile community samples and half of the motile fractions 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. 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. 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) using the original Earth Microbiome Project protocol using primers 515F and 806R (Caporaso et al., 2012), and with COI gene metabarcoding (for the macrobe community) using a 313 bp fragment of the COI gene with the primer set mICOIintf and jgHCO2198 (Geller et al., 2013; Leray et al., 2013). Additionally, the sessile and 100m-500m fractions were analysed with 18S rRNA gene metabarcoding (for macrobes) using a 536 bp fragment form the V4 region of the 18S rRNA gene with the primer set V4.18SNext.Fb and V4_18SNext.Rb primers (Appril et al., 2015). We selected primers that are widely used across metabarcoding studies, and which provide a broad representation on the gene regions targeted. PCR and sequencing protocols followed those in Casey et al., (2021).