Samples were acquired by the scientific drilling vessel JOIDES Resolution at IODP Site U1385A (37°34.285’N, 10°7.562’W) on November 25th, 2011 by Advanced Piston Coring (APC). Whole-round sections meant for molecular analysis were immediately frozen at -80 degrees C for the remainder of the cruise and shipped at this temperature. DNA was extracted using the MO BIO PowerMax Soil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) with a minor adjustment to manufacturer’s protocol. Briefly, 10 g of sediment extruded from the interior (>1cm from core liner) of whole-round core samples was processed according to manufacturer’s instructions, save for an additional step of incubation in a 65 degree C water bath for 15 minutes prior to step 4 (10 minute vortex). RNA was extracted using a modified protocol of the FastRNA Pro Soil-Direct Kit (MP Biomedicals, Solon, OH, USA) as described in Orsi et al. 2013b. 20 grams of thawed sediment was extracted from the center of each core whole-round sample and 5 grams placed in each of four 15 ml conical tubes. 5 mls of RNA soil lysis solution was added to each tube and vortexed for ~30 seconds. Additional soil lysis solution was added until ~ 1 ml of headspace remained. Tubes were then placed on a homogenizer (MP Biomedicals, Solon, OH, USA) and homogenized at a speed setting of 4.5 for 60 seconds. Two 15 ml tubes were combined into 50 ml conical tubes and centrifuged at 4000 rpm for 20 minutes. Supernatants were combined into one 50 ml tube. Roughly one- tenth volume of 2M sodium acetate pH 4.0 and 1 volume of phenol:chloroform pH 6.5 was added and samples were vortexed for 15 seconds and incubated at room temperature for 5 minutes. Samples were then centrifuged for 20 minutes at 4000 rpm. The top phase was removed to a new 50 ml tube. A roughly one-tenth volume of the kit’s inhibitor removal solution was added and samples were again centrifuged for 5 minutes at 4000 rpm. Supernatant was poured into a new tube and 2.5 volumes of 100% molecular grade ethanol and one-tenth volume of 3M sodium acetate was added. Tubes were then briefly vortexed and allowed to precipitate overnight at -80 degrees C.
The next day, samples were spun down for 60 minutes at 4000 rpm at 4 degrees C. An additional treatment with phenol:chloroform and inhibitor removal solution was performed before a 1 hour precipitation with 100% isopropanol at -20 degrees C. Samples were centrifuged at 14,000 rpm for 20 minutes and pellet was washed with 70% ethanol and centrifuged again. Pellet was allowed to air dry and was resuspended in 200 μl of RNAse free water. A one-tenth volume of Turbo DNAse buffer (Life Technologies, Carlsbad, CA, USA) and 1 ul of Turbo DNAse was added and incubated at 37 degrees C to get rid of contaminating DNA. Tubes were centrifuged at 14,000 rpm for 2 minutes and supernatant was aspirated into a new RNAse free 1.5 ml tube. The FastRNA Pro Soil-Direct kit instructions were then followed to the end, starting at step 18 (RNA matrix/slurry step) and including the optional “centrifugation through quick- clean filters” step. PCR using DNA polymerase with 2 ul of RNA as template was performed to check for left over contaminating DNA. No DNA-based PCR product was seen (data not shown). DNA extracts (including an extraction blank) were amplified using primers for the V4 hypervariable region of the eukaryotic 18S rRNA gene (Orsi et al. 2013a). PCR was performed for 35 cycles using SpeedSTAR HS DNA polymerase (Clontech Laboratories Inc., Mountain View, CA, USA) at an annealing temperature of 63 degrees C. Reactions were performed in three separate 25 ul aliquots and pooled. Reactions were checked by gel electrophoresis and product was gel-purified using the Wizard SV Gel and PCR Cleanup system (Promega Corporation, Madison, WI, USA). Concentrations were checked via Qubit v2.0 (Life Technologies, Carlsbad, CA, USA) and equimolar aliquots of product were sent to Molecular Research LP (Shallowater, TX, USA) for barcoding and sequencing on Illumina MiSeq platform per manufacturer’s instructions.
For RNA extracts (including an extraction blank), amplicons were generated with the Superscript One-Step RT-PCR system with Platinum Taq DNA polymerase (Life Technologies, Grand Island, NY, USA) using pre-barcoded primers for the V4 hypervariable region of the eukaryotic 18S rRNA gene (Orsi et al. 2013a, same core primers used for DNA amplicons). The cDNA synthesis was performed at 50 degrees C for 30 minutes, followed by 35 cycles of PCR amplification according to the manufacturer’s thermocycling protocol. Triplicate 50 ul RT-PCR reactions were pooled and checked for product via gel electrophoresis. RT-PCR products were gel purified using the QIAquick Gel Purification kit (Qiagen, Netherlands) and quantified via Qubit v2.0. Equimolar quantities of barcoded RT-PCR amplicons were pooled and sent for 454 pyrosequencing with FLX Titanium chemistry according to manufacturer’s instructions at Selah Genomics (Greenville, South Carolina, USA).