Mag bind rxnpure plus

Dust was collected at each site using a vacuum fitted with Dustream collectors (Indoor Biotechnologies, Charlottesville, VA). Collector filters (40-μm-pore mesh) containing the vacuumed dust were placed into sterile Nasco Whirl-Paks bags (Nasco, Fort Atkinson, WI) and stored under dark conditions at room temperature during collections. Collection took place from 18 July to 21 November 2016. Each sample was homogenized, and 0.25 g of dust was aliquoted into sterile 2-ml tubes and stored at −80°C until DNA extraction.
The DNA from dust aliquots was extracted using the MoBio PowerLyzer PowerSoil DNA isolation kit (MoBio, Carlsbad, CA, USA) protocol. To perform a metagenomic analysis, 1 ng of gDNA from each sample was prepared using the Illumina Nextera XT DNA library prep kit, along with the corresponding Illumina index kits v2 set A and set B, following the manufacturer’s instructions through the amplification step. Amplified products were purified with a modified bead-based DNA cleanup protocol using Mag-Bind RxnPure Plus by Omega Bio-Tek (Norcross, GA), quantified using the Quant-iT double-stranded DNA (dsDNA) assay kit, and pooled with equal concentrations of product using an Eppendorf (Hamburg, DE) epMotion 5075 robot. Libraries were sequenced on an Illumina HiSeq 4000 with 150-bp paired-end reads (insert size ranged from 250 to 1,000 bp).

DNA extractions were processed at the University of Connecticut’s Microbial Analysis, Resources, and Services facility for 16S rRNA gene amplifications and sequencing. Bacterial 16S rRNA genes were amplified using 30 ng of extracted DNA from each of the 192 soil samples. The V4 region was amplified using primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) with Illumina adapters and dual indices. Samples were amplified in triplicate using GoTaq (Promega) with the addition of 10 μg BSA (New England BioLabs), and the three independent PCR reactions were pooled prior to sequencing. The PCR reaction was incubated at 95∘C for 3.5 min, then 30 cycles of 30 s at 95.0∘C, 30 s at 50.0∘C and 90 s at 72.0∘C, followed by final extension as 72.0∘C for 10 min. PCR products were pooled for quantification and visualization using the QIAxcel DNA Fast Analysis (Qiagen). PCR products were normalized based on the concentration of DNA then pooled using the QIAgility liquid handling robot. The pooled PCR products were cleaned using the Mag-Bind RxnPure Plus (Omega Bio-tek) according to the manufacturer’s protocol. The cleaned pool was sequenced on the MiSeq platform using v2 2 × 250 chemistry (Illumina, Inc).

Genomic DNA was extracted from replicate samples or pooled replicates (Table S1, sampling replicates and sequencing replicates) using a DNeasy PowerFood microbial kit (Qiagen, Hilden, Germany). Extracted DNA samples were amplified and sequenced at the University of Connecticut Microbial Analysis, Resources, and Services Center using the standard protocol. DNA was quantified using Pico-Green fluorescent dye (Invitrogen, Waltham, MA, USA) and was used as the template to amplify the bacterial 16S rRNA V4 region (515F and 806R) and fungal internal transcribed spacer regions (ITS3 and ITS4). Primers with Illumina adapters and dual barcodes were used for amplification. PCR conditions for bacteria consisted of 94°C for 2 min, 30 cycles of 30 s at 94°C, 30 s at 53°C, and 60 s at 72°C, and a final extension at 72°C for 5 min. PCR conditions for fungi consisted of 95°C for 2 min, 5 cycles of 30 s at 95°C, 60 s at 48°C, and 60 s at 72°C, 25 cycles of 30 s at 95°C, 60 s at 55°C, and 60 s at 72°C, and a final extension at 72°C for 5 min. PCR products were cleaned using Mag-Bind RxnPure plus (Omega Bio-tek, Norcross, GA, USA) and sequenced on Illumina MiSeq using the 2 × 250-bp kit (Illumina, Inc., San Diego, CA, USA). Negative extraction controls were also sequenced to test for contamination.