Page 204 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 9. Long-term warming effects on permafrost soil microbial communities
maximum time interval of 140 days. GHG production rates was calculated by a moving window for every four measurements, and displayed as μg per day per gram soil organic carbon (μg d- 1 g SOC-1).
DNA extraction, qPCR and sequencing
DNA was extracted from both pre-incubation and post-incubation samples. Before DNA extraction, plant residues were removed step-by-step by using sterilized sieves (2 mm, 1 mm and 0.5 mm) in slurries with 1x phosphate-buffered saline (PBS) under aseptic conditions. The PBS flow-through was pooled with the remaining material (after sieving), and the material was filtered through a 0.2 μm pore size filter. The total genomic DNA was extracted on the filter by using a phenol-chloroform method (Tveit et al. 2015). After RNA removal with RNaseA (0.5 μl, 10 mg mL-1 per reaction) at 4°C, the obtained DNA was sent for paired-end sequencing at GATC Biotech, Germany (now Eurofins Scientific, Germany) on an Illumina Hiseq 2500 system.
Quantitative PCR. qPCR was conducted by using a CFX Real-Time PCR Detection System (Bio-Rad Laboratories). The primer sets Eub341-F/Eub534-R (Muyzer et al. 1993) and mlas/mcrA-rev (Steinberg and Regan 2008) were used to target bacteria and methanogenic archaea, respectively. The reaction solutions contain iTaqTM Universal SYBR Green Supermix (Bio-Rad Laboratories), 0.5 μM primer each, and 2 μL diluted (1:100) template DNA. The qPCR assays contain the following steps: initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation for 3 s at 95°C, annealing for 20 s at 58.5°C, elongation for 30 s at 72°C, and a plate read step at 80°C for 0.3 s. Melt curve analysis from 65-95°C with a 0.5°C temperature increment per 0.5 s cycle was conducted at the end of each run. Based on standard curves, the PCR efficiencies ranged between 93% and 99%.
Metagenome sequencing & analysis. In total, 89 Gb of metagenomic sequence data were generated (70.8-84.5 million sequences per sample). Raw metagenomic sequences were processed with the ATLAS metagenomic pipeline (v2.0.6) (Kieser et al. 2019) by following the standard procedure. Briefly, the workflow includes quality control, assembly (metaSPADes was used in this study), genome binning and annotation (for details please refer to [Kieser et
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