Page 179 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

methanogens and for biotic controls without any substrate amendment. Samples were incubated at 4°C and 10°C. Substrates were replenished when CH4 production leveled off. For acetate amended cultures a total of 14 mM acetate was added (pulses of 4 mM at day 64 and 2 mM at day 0, 141, 190, 211 and 234) and 12 mM of TMA (pulses of 2 mM at day 0, 141, 190, 211, 234 and 269) during the 279 days of incubation.
DNA extraction & sequencing
DNA isolation. Sediment samples were taken aseptically and pelleted by centrifugation for 10 min at 20,000 x g. Pellets were stored at -18°C until DNA isolation. Samples for DNA analysis were obtained by pooling equal amounts of pelleted slurry sample of each triplicate incubation. DNA was extracted in duplicate per sample using two different extraction methods. For the first method DNA was extracted using the PowerSoil DNA Isolation Kit (MO BIO, Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions with the following modifications. PowerBead Tubes were inserted in a TissueLyser LT (Qiagen, Venlo, the Netherlands) at 50 Hz. DNA was eluted from the spin column in two elution steps with 2x 25 μL sterile Milli-Q. DNA samples were stored at -18°C until further analysis.
For the second method DNA was extracted using the cetyl-trimethyl-ammoniumbromide (CTAB) extraction buffer protocol as described by Zhou, Bruns and Tiedje (1996). For the final step DNA pellets were resuspended in 40 μl sterile DEPC-treated Milli-Q by pipetting and incubation overnight at 4°C. NanoDrop analysis indicated contamination of DNA samples with organics. DNA samples were purified using Agencourt AMPure XP beads following the manufacturer’s instructions (Beckman Coulter, Brea, CA). DNA quality was checked by agarose gel electrophoresis and spectrophotometrically using the NanoDrop 1000 (Invitrogen, Thermo Fisher, Carlsbad, CA). DNA quantity was measured fluorometrically by using the Qubit dsDNA HS Assay Kit (Invitrogen, Thermo Fisher, Carlsbad, CA) according to the manufacturer's instructions.
Metagenome sequencing. Library preparation of the metagenomes (one library per DNA extraction for each metagenome) was done using the Nextera XT kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. Enzymatic tagmentation was performed with 1 ng input DNA, followed by incorporation of the indexed adapters and amplification of the library.
8
177