of extraction buffer (50 mM sodium acetate and 10 mM EDTA, pH 5), and 0.5 ml 20% SDS. This mixture was bead-beaten with 0.1 mm silica beads, then extracted sequentially with phenol, phenol-chloroform (1:1), and chloroform, precipitated in 7.5 mM ammonium acetate mixed with isopropanol, and washed with 80% ethanol. The pellet was resuspended in water and incubated with 4 ml TurboDNase at 37uC for 30 minutes, followed by purification with the Qiagen RNeasy MinElute kit. Bacterial 16S rRNA cDNAs were amplified with B8f-B1492r [47] with an annealing temperature of 60uC for Mat-B (0–3 cmbsf), Mat-B (12–15 cmbsf), and Edge (0–3 cmbsf) and 58uC for the rest; dsrAB transcript cDNAs were amplified with DSR1f- DSR4r [87] at an annealing temperature of 54uC with a nested reamplification with 1f1r [50] at 48uC; and mcrA transcript cDNA were amplified with ME1-ME2 [61] at an annealing temperature of 55uC. For amplification of archaeal 16S rRNA genes, A8f and A1492r [47] were used at an annealing temperature of 59–60uC. In the samples from outside the mat, initial amplification using these primers had to be followed by nested reamplification with primer combination A21f-A915r [88,89] at an annealing temper- ature of 58uC in order to see a product on a 1.5% agarose gel. Primer sequences and known mismatches are listed in Table S2. All reverse transcription and PCR reactions took place in a single tube using the reverse primer as the reverse transcription primer. Each 25 ml RT-PCR reaction contained 1 ml RNA template, 0.15 ml each primer solution (100 pmol/ml), 1 ml bovine serum albumin (10 mg/ml; absent in bacteria reactions), as well as the following products from the Takara OneStep RT-PCR kit Version 3.0: 12.5 ml buffer, 0.5 ml RNase inhibitor, 0.5 ml HotStar Taq, and 0.5 ml reverse transcriptase. Each 25 ml nested PCR reaction contained 1 ml cDNA template, 0.15 ml each primer solution (100 pmol/ml), 1 ml bovine serum albumin (10 mg/ml), 4 ml deoxynucleotide triphosphate (10 mM each dATP, dCTP, dGTP and dTTP), 2.5 ml 106 FastBuffer I (Takara), and 0.125 ml SpeedStar Taq (Takara). Conditions for RT-PCR in a Bio-Rad iCycler (Hercules, CA) were as follows: reverse transcription at 42uC for 15 min, reverse transcriptase inactivation and polymerase activation at 95uC for 2 min, followed by 25 cycles for bacterial 16S rRNA cDNA and archaeal 16S rRNA cDNA and 40 cycles for dsrAB mRNA cDNA and mcrA mRNA cDNA, each consisting of 5 s denaturation at 95uC, 15 s at primer annealing temperature (see above), and 20 s elongation at 72uC, plus a final elongation at 72uC for 10 min. Nested PCR for dsrAB required the following protocol: 94uC polymerase activation for 2 min, followed by 40 cycles of 98uC denaturation for 10 s, 48uC annealing for 15 s, and 72uC extension for 20 s, plus a final elongation at 72uC for 10 min. All PCR and RT-PCR products were purified using either a MoBio PCR Clean-up kit or purification in a 1% agarose gel and MoBio UltraSpin for gel purification. Purified products were cloned using the TOPO TA PCR cloning Kit, and transformed into E. coli by electroporation following the manufacturer’s protocols (Invitrogen, San Diego, California). Sequences were obtained at the Josephine Bay Paul Center at the Marine Biological Laboratory (Woods Hole, MA), using an ABI 3730 sequencer, or at Genewiz (South Plainfield, NJ) on an ABI Prism 3730xl sequencer. Vector and primer sequences were removed from sequences and forward and reverse reads were assembled into contigs using Sequencher 4.7. Ribosomal sequences were aligned against the 2007 Silva release with ARB (www.arb-home. de). Sequences were deposited in NCBI Genbank with accession numbers GU190968-GU191015 for archaeal 16S, GU302419- GU302497 for bacterial 16S, GU302498-GU302509 for mcrA, and GU302510-GU302521 for dsrAB. Patterns of CH4-Seep RNA Tag Sequencing DNA was extracted from 0.5 g of sediment using the MoBio DNA Power Soil Kit (MoBio Inc, Carlsbad, CA). Using the methods of the International Census of Marine Microbes (ICoMM), the variable 6 (V6) region of the 16S rRNA gene was amplified and subjected to 454 pyrosequencing on a Roche GS20. All PCR methods, primers and analysis tools are detailed on the ICoMM website (www.vamps.mbl.edu; see also 17). Quality control included removing sequences with ambiguous base calls, or ones that did not match the primers perfectly [90]. Chao1 estimates are shown at 3% OTU clustering, therefore insertions and deletions of individual bases during amplification or pyrosequencing did not contribute to diversity estimates. Tag sequences are publicly available from http://vamps.mbl.edu as the following datasets: GMS_0003_2006_09_14 (bacteria, under mat), GMS_0004_2006_09_14 (archaea, under mat), GMS_0005_ 2006_09_14 (bacteria, outside mat) and GMS_0006_2006_ 09_14(archaea, outside mat). Controls on RT-PCR Reverse transcriptase-free control RT-PCR reactions were made for each clone library to check for the co-extraction of DNA. No PCR products were visible on a 1.5% agarose gel for any of the controls. In order to check for PCR products not visible in the gel, nine No RT controls from six different RNA extractions were gel purified, cloned, and sequenced. Less than 10% of plasmids contained any inserts, and of those that did, most were plasmid DNA or other bits of DNA not present in any of the RT-PCR clone libraries. Three clone libraries contained 4 clones total of Eel-2 Bacterial 16S rRNA gene sequences identical to the most numerous clone in RT-PCR clone libraries. However, since 1) these PCR products were gel purified alongside concentrated RT-PCR products used to guide the cutting of the invisible bands, and 2) two of the three No RT clone libraries were made with dsrAB primers, not bacterial 16S rRNA ones, it is likely that this small number of sequences were contamination from RT-PCR products during gel- cutting. Extraction blanks were also carried through all stages of RNA extraction, purification, RT-PCR, and nested PCR, where appro- priate. No extraction blanks were visible on gels for any clone library. Sequence Analysis Operational taxonomic units (OTUs) were determined by aligning 500–600bp of each forward read in ClustalX, and grouping into 99% similar OTUs using a distance matrix generated in PAUP4.b10 [91]. Representatives of each OTU were reverse sequenced to get a full-length read. Chimeras were identified using Pintail and also by Blasting 59 and 39 ends separately to check for agreement. Full-length and short reads were then aligned using ARB (www.arb-home.de), and phyloge- netic groups were determined. Only full-length reads were included in the phylogenetic trees, which were made in PAUP. Chao1 diversity estimates were calculated using the methods of DOTUR [92], which are based on the EstimateS modification [93] of the original Chao1 diverisity estimator [94]. Chao1 values (SChao1) were calculated with a bias correction for the presence of singletons as SChao1 = Sobs+n1 (n121)/(2*(n2+1)), where Sobs is the observed number of species, n1 is the number of OTUs with only one sequence, and n2 is the nmber of OTUs with only two sequences. Chao1 is a method for predicting actual diversity, assuming that only a subset of the total population has been sampled; and works well at a low average sample capture probability [95]. All samples except for archaea Edge 21–24 cmbsf and bacteria Edge 0–3 cmsf and Edge 12–15 cmbsf deviated PLoS ONE | www.plosone.org 10 January 2010 | Volume 5 | Issue 1 | e8738