The frozen samples from series D of batch e previously incubated at 3, 4, 5, 14, 15, 16, 24, 25, and 26 °C were homogenized separately with mortar and pestle in liquid nitrogen. Coextraction of RNA and DNA from peat soil was performed as previously described (20 (link)). Briefly, a cetrimonium bromide-containing lysis buffer and phenol:chloroform:isoamylalcohol (25:24:1) were added to all peat samples in lysis matrix E tubes (MP Biomedicals) containing silica beads and exposed to 30 s of vigorous shaking in a FastPrep machine (MP Biomedicals) for the extraction of nucleic acids. After PEG precipitation, ethanol washing and dissolution of pellets in nuclease-free water, nucleic acids were treated with DNase or RNase before metatranscriptome and metagenome generation, respectively. Total RNA was amplified using the MessageAmp II-Bacteria Kit (Ambion Life Technologies) following the kit protocol, except that the linear amplification step was carried out for 14 h. Paired-end 101-bp reads were sequenced using the Illumina HighSeq2000 sequencer (Illumina) at the Norwegian Sequencing Centre (University of Oslo, Oslo, Norway).
Protocol detail
Peat Soil Nucleic Acid Coextraction
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Bacteria
Buffer
Cetrimonium bromide
Chloroform
Dnase
Ethanol
Frozen
Metagenome
Nitrogen
Nucleic acids
Pellets
Phenol
Rnase
Silica
Corresponding Organization :
Other organizations : UiT The Arctic University of Norway, University of Vienna, Austrian Polar Research Institute, Max Planck Society
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Variable analysis
independent variables
- Incubation temperature (3, 4, 5, 14, 15, 16, 24, 25, and 26 °C)
- Nucleic acid (RNA and DNA) extraction and purification
- Batch e of frozen samples from series D
- Homogenization of samples in liquid nitrogen using mortar and pestle
- Cetrimonium bromide-containing lysis buffer and phenol:chloroform:isoamylalcohol (25:24:1) for nucleic acid extraction
- Lysis matrix E tubes (MP Biomedicals) containing silica beads
- FastPrep machine (MP Biomedicals) for vigorous shaking
- PEG precipitation, ethanol washing, and dissolution of pellets in nuclease-free water
- DNase or RNase treatment before metatranscriptome and metagenome generation, respectively
- MessageAmp II-Bacteria Kit (Ambion Life Technologies) for RNA amplification
- 14 h linear amplification step
- Paired-end 101-bp reads sequenced using the Illumina HighSeq2000 sequencer (Illumina) at the Norwegian Sequencing Centre (University of Oslo, Oslo, Norway)
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