Metagenomic DNA was extracted with three different techniques each from a 0.5 g soil sub-sample and pooled (De León-Lorenzana et al., 2017 (link)). As such, 1.5 g soil from each plot (n = 3), treatments (n = 12) and sampling day (n = 5) was extracted for metagenomic DNA. Overall, 270 g soil was extracted and 180 DNA samples were obtained.
The V3-V4 hypervariable regions of 16S rRNA genes were amplified with DNA polymerase (Thermo Fisher Scientific, Hudson, NH, USA) in a final 25 μl reaction volume. Amplification was done with the PCR Thermal Cycler Multigene Optimax (Labnet, Edison, NJ, USA). The following thermal cycling scheme was used: initial denaturation at 94 °C for 10 min; 25 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 10 min, using the forward primer 341F (5′-CCTACGGGIGGCWGCAG-3′) and the reverse primer 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing the Illumina platform adapters and eight bp barcodes. All samples were amplified in triplicate, pooled in equal volumes and sequenced by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea) with paired-end Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA).
The V3-V4 hypervariable regions of 16S rRNA genes were amplified with DNA polymerase (Thermo Fisher Scientific, Hudson, NH, USA) in a final 25 μl reaction volume. Amplification was done with the PCR Thermal Cycler Multigene Optimax (Labnet, Edison, NJ, USA). The following thermal cycling scheme was used: initial denaturation at 94 °C for 10 min; 25 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 10 min, using the forward primer 341F (5′-CCTACGGGIGGCWGCAG-3′) and the reverse primer 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing the Illumina platform adapters and eight bp barcodes. All samples were amplified in triplicate, pooled in equal volumes and sequenced by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea) with paired-end Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA).
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