Dataset: ICoMM GPS
Deployment: AT11-04

Filtration
Water samples were pre-screened through 200 µm Nitex mesh followed by 80 µm Nitex mesh
(to reduce microbial metazoa e.g., larval stages, copepods, etc.) directly from Niskin
sampling bottles.  Microbial biomass in the < 80 µm size-fraction was collected onto GF/F
filters at low vacuum pressure  (< 10 mm Hg) and stored with 2 ml of lysis buffer (as in
Countway et al., 2005) at -80°C until extraction.  Sediments were retrieved from the seafloor
cores, sectioned on board ship at one centimeter intervals, and stored at -80°C until processing
in our home laboratories.  Samples from microbial mats were not pre-screened prior to freezing
at -80°C.

Molecular processing
High molecular weight DNA was extracted from sediment samples using the MoBio power soil
kit (MoBio, Carlsbad, CA. USA).  DNA from water samples collected on GF/Fs was extracted
using a standard Phenol:Chloroform:Isoamyl alcohol method and precipitated with 95% ethanol. 
All extracted samples were quantified on a Nanodrop 1000 (University of Delaware) or by
fluorometry (University of Southern California) using PicoGreen (Invitrogen).  DNA samples
for eukaryote ‘pyrotag sequencing’ were sent to the MBL in Woods Hole, MA for amplification
by PCR and subsequent library preparation as described in Amaral-Zettler et al. (2009).
Preliminary taxonomic assignments were made for all pyrotag sequences using the ‘GAST’
approach described in the previous reference.

Amaral-Zettler, L.A., McCliment, E.A., Ducklow, H.W., and Huse, S.M. (2009) A Method for Studying
Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit
Ribosomal RNA Genes. PLoS ONE 4: e6372. doi:6310.1371/journal.pone.0006372.

Countway, P.D., Gast, R.J., Savai, P., and Caron, D.A. (2005) Protistan diversity estimates based
on 18S rDNA from seawater incubations in the western North Atlantic. Journal of Eukaryotic Microbiology 52: 95-106.