Maize LDPE Macro 25 Year
16S gene sequencing was conducted for each treatment (n = 3-5). At the end of the experiment, soil samples (ca. 15 g) were collected from the root zone of each treatment and stored at -80C before freeze-drying. Bacterial DNA was extracted from each sample using the Zymo BIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA) according to manufacturers instructions. Two independent DNA extractions were carried out with a high-speed bead beating for each sample. Quality and concentration of extracted DNA were assessed by gel electrophoresis and by Qubit 4.0 Fluorometer dsDNA BR Assay Kit (Life Technologies, Carlsbad, CA). Libraries of 16S rRNA gene amplicons were prepared by single PCR with double-indexed fusion primers as described previously (Fadrosh et al., 2014). Hypervariable V4 16S rRNA gene fragment was amplified using modified forward primer F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 prokaryotic primer (5-GGACTACHVGGGTWTCTAAT-3), which amplify an approximately 290 bp region. Primers were designed to contain: the Illumina adapters and sequencing primers, a 12 bp barcode sequence, a heterogeneity spacer to mitigate the low sequence diversity amplicon issue, and 16S rRNA gene universal primers (Fadrosh et al., 2014). PCRs were performed using OneTaq DNA Polymerase (New England Biolabs, Ipswich, MA). All reactions were run with no-template negative controls. Thermocycling conditions were: initial denaturation at 95C for 2 min, followed by 30 cycles at 95C for 30 s, 50C for 50 s, and 72C for 90 s with a final elongation at 72C for 5 min. Amplicons were visualized in a 1.5% tris-acetate agarose gels using a GelDoc System (Bio-Rad, Hercules, CA). DNA bands of approximately 440 bp were gel-purified using QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). The purified amplicons were then quantified using Qubit 4.0 Fluorometer, pooled in equimolar amounts and the final pool was run on Illumina MiSeq platform (Illumina Inc., San Diego, CA) using 500-cycle v2 chemistry (2  250 bp paired-end reads) at the Centre for Environmental Biotechnology, Bangor, United Kingdom.