Crustal fluids were collected from the single horizon at U1382A and from the shallow, middle, and deep horizons at U1383C using an ROV-based pumping and filtration system tailored for microbial sampling. The mobile pumping system (or MPS) is described in:
Cowen, J. P. et al. 2012. Advanced instrument system for real-time and time-series microbial geochemical sampling of the deep (basaltic) crustal biosphere. Deep-Sea Research Part I: Oceanographic Research Papers 61, 43-56, doi:10.1016/j.dsr.2011.11.004

Individual CORK fluid delivery lines were flushed using the MPS at a rate of ~4 liters per minute for at least 3 times volume of the fluid delivery line (~20-30 minutes) prior to diverting fluid flow to six 15 L foil-lined sample bags (Jensen Inert Products) that were acid cleaned and sterilized using gamma irradiation prior to deployment. Fluids were not sampled until at least 15-30 minutes of stable, reproducible measurements were observed, indicating a fully flushed fluid delivery system and access to crustal fluids. In addition, ~5 L of fluid from each of the three horizons in U1383C was filtered in situ onto 47 mm SUPOR filters in pancake-style filter holders (McLane Inc). These filtered samples were preserved in situ using a reservoir of RNA Later (Qiagen) that is part of the MPS pumping system. Once recovered, ten liters of each sample was filtered onto a 0.22 um Sterivex-GP filter at 5 degrees C for microbial analysis. Similarly, bottom seawater was collected by CTD at 100 m above the sea floor and filtered in the same manner. In situ and ship-based filters were fixed at 4 degrees C for 18 hours with RNA Later immediately after filtering or upon recovery, then frozen at -80 degrees C until nucleic acid extractions. B. Orcutt provided a frozen sample of drilling mud from U1382A from IODP Exp. 336.

Sterivex filters and 47 mm flat filters were cut into two equal pieces using sterile technique. Total genomic DNA was extracted from one half using a phenol chloroform method as previously described in:
Sogin, M. L. et al. 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proceedings of the National Academy of Sciences of the United States of America 103, 12115-12120, doi:10.1073/pnas.0605127103

RNA was extracted from the other half with a mirVana miRNA isolation kit (Ambion Inc) preceded by a bead beating step using RNA Powersoil beads (MoBio). Extracted RNA was treated with Turbo DNase (Ambion Turbo DNA-free kit) and converted to cDNA with an Applied Biosystems (ABI) High Capacity RNA to cDNA kit prior to amplicon library preparation. Total genomic DNA was extracted from approximately 1 g of drilling mud using a MoBio UltraClean® Soil DNA Isolation Kit.

Whole crustal fluids and bottom seawater were fixed with 3.7% formaldehyde for cell counts. Up to 19.8 ml of fixed fluids were filtered onto a 0.2 um GTBP polycarbonate filter (Millipore Inc), stained with DAPI (4′,6′-diamidino-2-phenylindole; Sigma), and counted via epiflourescent microscopy. For fluorescence in situ hybridization (FISH), cells were filtered onto 0.2 um GTTP polycarbonate filters (Millipore Inc) and fixed with 2% paraformaldehyde, rinsed with milliQ H2O, air dried and stored at –20 degrees C until further use. Cells on filters were hybridized with HRP-labeled 16S rRNA targeted oligonucleotide probes EUB338 21, ARCH915 22 and NON338 21 (Biomers GmbH, Ulm, Germany), and the signal was amplified using Alexa 488® tyramides (Invitrogen). The permeabilization step of the protocol before probe hybridization was modified, such that the cells on the filters were first permeabilized with Proteinase K (0.005 U ul –1 in 0.05 M EDTA, 0.1 M Tris-HCl, at pH 8) for 30 minutes at 37 degrees C. Filters were then washed in 50 ml 1X PBS at room temperature, followed by a second permeabilization treatment with Lysozyme (106 U ml–1, in 0.05 M EDTA, 0.1 M Tris-HCl, at pH 8) for 30 minutes at 37 degrees C. After signal amplification, all cells were counterstained with DAPI and counted via epiflourescent microscopy.

The relative abundance of bacterial and archaeal 16S rRNA genes was determined by qPCR assays as previously described (Nadkarni, M. a., Martin, F. E., Jacques, N. a. & Hunter, N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148, 257-266, doi:10.1128/JCM.40.5.1698 (2002) and Takai, K. & Horikoshi, K. Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes. Applied and environmental microbiology 66, 5066-5072, doi:10.1128/AEM.66.11.5066-5072.2000). To generate standards, plasmid DNA was extracted from Axial Seamount low-temperature diffuse vent clone libraries, purified, and linearized using the WizardPlus SV Minipreps DNA Purification System (Promega Inc). Standards were constructed by mixing equal amounts of four bacterial plasmids for the quantification of bacterial 16S rRNA gene and two archaeal plasmids for the quantification of archaeal 16S rRNA gene. A 1:10 dilution series of the plasmid mixtures beginning with an initial concentration of 0.06 ng/ul (bacteria) and 0.10 ng/ul (archaea) was used to produce standard curves with R2 values > 0.991 and with efficiency ranging from 93 to 96%. Each 20 ul reaction contained KAPA PROBE FAST ABI Prism® 2X qPCR Master Mix (Kapa Biosystems Inc), forward and reverse primers at optimized concentrations of either 3 nM (bacteria) or 4 nM (archaea), optimized probe concentrations of either 2.5 nM (bacteria) or 5.0 nM (archaea), DEPC-treated water, and 2 ul of DNA template. Triplicate reactions were performed on a StepOne Plus Real Time PCR System (Applied Biosystems Inc) for each sample and for no template controls. Cycles began with initial denaturation for 3 min at 96 degrees C, followed by 40 cycles of 15 s at 96 degrees C and 3 min at 59 degrees C. STEPONE software version 2.2.2 (Applied Biosystems Inc) was used to analyze the results.