Dataset: NP_16SrRNA_Huber
Deployment: MSM20-5

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. The V6 region of 16S rRNA genes was amplified in triplicate for each sample with previously reported primers designed for archaea and bacteria (Huber, J. A. et al. Microbial population structures in the deep marine biosphere. Science (New York, N.Y.) 318, 97-100, doi:10.1126/science.1146689 (2007)) that were modified to include indices and barcodes compatible with the Illumina HiSeq1000 platform rather than 454 Life Sciences Adapters (Eren, A. M., Vineis, J. H., Morrison, H. G. & Sogin, M. L. A Filtering Method to Generate High Quality Short Reads Using Illumina Paired-End Technology. PLoS ONE 8, 6-11, doi:10.1371/journal.pone.0066643 (2013)). Triplicate PCR amplifications were pooled for each sample, cleaned with a Qiagen MinElute kit, and quanitified by PicoGreen assay on a Turner Biosystems spectrophotometer. Fifty nanograms of each cleaned amplicon library was then size selected with a 2% agarose PippinPrep cassette to produce a narrow range of fragment sizes from 200 to 240 bp for sequencing and cleaned again to remove agarose. Equimolar amounts of pooled amplicon libraries and a metagenomic library were run in the same lane to avoid known difficulties of sequencing low-complexity amplicon libraries with Illumina (Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal 6, 1621-1624, doi:10.1038/ismej.2012.8 (2012)).