Page 83 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

DNA extraction
After 17 and 25 months, DNA was extracted from 2 g of well sediment using the phenol/chloroform extraction method as described by Lueders et al. (2004). Subsequently, DNA was precipitated using polyethylene glycol 6000 (Sigma-Aldrich, St Louis MI, United States), and the DNA pellet was washed once with 70% ethanol and dissolved in 50 μl elution buffer (Qiagen, Venlo, the Netherlands). Three parallel extractions were carried out, and extracts were pooled for each incubation treatment. DNA concentration and purity were determined by standard agarose gel electrophoresis and fluorometrically using RiboGreen assays (Qubit Assay Kit, Invitrogen, Waltham MA, United States) according to the manufacturer’s instructions.
DNA sequencing and data processing
Both the 17- and 25-month DNA samples were used for 16S rRNA gene amplicon sequencing on the IonTorrent PGMTM. Amplification of the V3-V4 region of the bacterial 16S rRNA gene was performed using universal primers Bac F341 (5’-CCTACGGGNGGCWGCAG-3’) and Bac785R (5-‘GACTACHVGGGTATCTAATCC-3’) (Klindworth et al. 2013) for 25 cycles. Archaeal 16S rRNA genes were amplified with the universal archaeal primers Archf349 (5’- GYGCASCAGKCGMGAAW-3’) and Archr789 (5’-GGACTACVSGGGTATCTAAT-3’) (Klindworth et al. 2013) for 30 cycles. Both PCR amplifications were performed by 10 min 98°C initialization, 25/30 cycles of 1 min denaturation at 95°C, 1 min of annealing at 60°C, 2 min of elongation at 72°C and a 10 min final elongation step at 72°C. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Venlo, the Netherlands) in two elution steps. 20 μl 55°C Milli-Q was added to the spin column and incubated for 2 min prior to centrifugation. Next, the eluate was put onto the spin column, incubated at 55°C for 2 min and centrifuged again as described in the manual. A 10-cycle nested PCR with IonTorrent adapters was performed on the purified PCR products using the same PCR protocol. After PCR purification with the QIAquick PCR Purification Kit as described above, PCR products were used for library preparation and sequencing steps according to the manufacturer’s instructions (Life Technologies, Carlsbad CA, United States). Amplicon sequences were quality checked for chimeras and clustered into OTUs with a 97% identity cut-off value using the 454 SOP (http://www.mothur.org/) (Schloss et al. 2009) with IonTorrent modified protocols. Chimeras
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