JGI-42989
16S rRNA gene amplicons from untreated peat soil, used to set up microcosm incubations       [DNA sample J1]
Excerpt from PMID 27015005: Peat soil microcosms were set up by mixing 30 g of fresh soil (10-20 cm depth) with 60 ml of filter-sterilized and anoxic peat water in 250 ml sterile glass bottles. Microcosms were sealed under 100% N2 with butyl rubber septa and incubated for 53 days in the dark at 14 °C. Microcosms were periodically amended from the first day of incubation with formate, acetate, propionate, lactate, or butyrate, or incubated without any external substrate. Half of the substrate-amended and -unamended microcosms additionally received periodic amendments of sulfate. These microcosms were initially spiked with sulfate, and, thereafter, were periodically amended with small amounts of sulfate. Triplicate microcosms were set up per incubation condition (36 microcosms in total). After 0, 5, 26 and 50 days of incubation, total nucleic acids were extracted from soil samples, purified, separated into RNA and DNA fractions and quantified: Soil samples were ground in liquid N2 and separated into approx. 300 mg aliquots, followed by total nucleic acids extraction as described previously (Leininger et al., 2006). Nucleic acids were purified with the OneStep PCR inhibitor removal kit (Zymo Research) and split into two fractions. Each fraction was either treated with RNase (RNase ONE ribonuclease, Promega) followed by sodium acetate precipitation (Sambrook and Russell, 2001) or DNase (TURBO DNA-free kit, Thermo Fisher Scientific) to obtain RNA-free DNA or DNA-free RNA, respectively. Complete removal of DNA in RNA samples was verified with a quantitative PCR (qPCR) assay targeting the 16S rRNA genes of most Bacteria and Archaea (Pester et al., 2010). PicoGreen dsDNA and RiboGreen RNA assays (Thermo Fisher Scientific) were used for nucleic acids quantification. cDNA was obtained using the SuperScript III first-strand synthesis kit (Thermo Fisher Scientific) with random hexamer primers for subsequent amplicon sequencing. The V4 region of the 16S rRNA gene and its cDNA was amplified as described previously (Caporaso et al., 2011) and sequenced in three Illumina MiSeq runs at the Joint Genome Institute (genome portal projects 1016201, 1016203, and 1031338). Reliability of relative abundance shifts in this amplicon sequencing approach was verified by a mock community analysis as internal control (Herbold et al., 2015).
M1020.H2
JGI
JGI-42989