Computational protocol: Fe-oxide grain coatings support bacterial Fe-reducing metabolisms in 1.7−2.0 km-deep subsurface quartz arenite sandstone reservoirs of the Illinois Basin (USA)

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Protocol publication

[…] Genomic DNA was extracted from the filter membranes, through which freshly collected formation water was filtered as introduced in the Supplementary Information. The conical tubes containing membranes in RNAlater were thawed on ice before centrifuged at 9000 rpm at 4°C for 45 min. The RNAlater supernatant was carefully removed and the filter membranes were blotted on sterilized Kimwipes®. The protocol for DNA extraction was modified from a previously published protocol (Tsai and Olson, ). Briefly, a lysis buffer containing pyrophosphate (He et al., ) and lysozyme (15 mg/mL) was used to minimize absorption of DNA on native iron minerals. This was followed with another incubation in STS solution (0.1 M NaCl, 0.5 M Tris-Cl, pH = 8.0, 10% sodium dodecyl sulfate) (He et al., ) and freeze-thaw cycling between a 55°C water bath and liquid nitrogen to further disrupt cell membranes for three times. After sequential phenol, phenol:chloroform (5:1) and phenol:chloroform:amyl alcohol (25:24:1) extractions, the supernatant was mixed with two volumes of Solution 3 of Ultraclean Soil DNA Isolation Kit (Mobio Laboratories, Inc., CA). The mixed solution was loaded on a silica spin filter and centrifuged at 10,000 × g. The filtrate was discarded and this step was repeated until all the mixed solution was filtered. The following DNA wash and elution was performed following the manufacture's recommendation. Using silica filters to collect DNA circumvented co-precipitation of DNA and salts when classical alcohol precipitation was applied, which might be due to the high salt concentrations in the samples. Unless mentioned, a negative control under the same extraction process was prepared to ensure no contamination was introduced during DNA preparation.In order to extract DNA from the active enrichment cultures, about 3 mL of well-mixed culture developed from the formation water samples 5655 and 6634 (referred to as IBDP5655 and IBDP6634 afterwards, respectively) was withdrawn with a N2 flushed syringe and transferred into microcentrifuge tubes. The samples were centrifuged at 13,000 rpm at 4°C for 10 min and the supernatant was discarded. DNA was extracted from the pellets by using FastDNA® Spin Kit for Soil (MP Biomedicals, CA).Microbial community composition in the formation water samples and active enrichment cultures were characterized using 16S rRNA clone libraries. Primers used for amplifying 16S rRNA genes were the Bacteria-specific primers 8F and 1492R (Liu et al., ) and Archaea-specific primers 21F and 958R (Elshahed et al., ). PCR was conducted with TaKaRa Ex Taq polymerase (TaKaRa Bio USA, Madison) using an Eppendorf MasterCycler (Eppendorf, Germany) following the recommendation of the manufacturers. The PCR products were verified by using agarose gel electrophoresis and then purified with QIAquick PCR Purification Kit (QIAGEN Inc., CA). Purified 16S rRNA genes were cloned into p-GEMT Easy Vector® and transformed into JM109 high efficiency competent cells as recommended by the manufacturer (Promega Corporation, WI). For each 16S rRNA clone library, a total of 192 recombinant plasmids were extracted from randomly picked E. coli colonies containing cloned sequences. The cloned insert in each plasmid DNA sample was amplified and sequenced using M13 primers at the Illinois Biotechnology Center of University of Illinois Urbana-Champaign, Urbana.The 16S rRNA gene sequences from the clone libraries were assembled with Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, Michigan). Sequences with low quality (e.g., not able to be assembled, ambiguity > 5 or with trimmed ends) were eliminated. Alignments and distant matrices of assembled sequences were generated with the aid of the NAST package in Greengenes (DeSantis et al., ). Chimeric artifacts were determined with Bellerophon (version 3) (Huber et al., ) and removed. Operational taxonomic units (OTU) were designated at sequence similarity levels of 97% with Mothur 1.28.0 (Schloss et al., ). Taxonomic classification was determined by sequence classification program from the Greengenes (DeSantis et al., ) and confirmed with Ribosomal Database Project (RDP) II database (Maidak et al., ). A neighbor-joining phylogenetic tree was constructed with the aid of ARB based on Jukes-Cantor correction (Ludwig et al., ; Tamura et al., ). The robustness of the inferred tree topologies was evaluated after 1000 bootstrap replicates of the neighbor-joining data. […]

Pipeline specifications

Software tools Sequencher, mothur
Databases Greengenes
Application Phylogenetics
Diseases Inert Gas Narcosis
Chemicals Hydrogen, Iron, Quartz, Pyruvic Acid