Zymoclean™ Gel DNA Recovery Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.	Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.
Protocol tips
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Publication protocol

Immediately prior to dosing, a portion of freshly prepared HE inoculum samples was collected and stored in −80 °C, at days 0, 2, 4 and 6. A total of 16 inoculum samples were collected, as four biological replicates were retrieved at each time point. To prepare for DNA extraction, frozen inoculum samples were thawed in a room temperature water bath. DNA was extracted using an adaptation of the methods described previously56,57. In short, cells were pelleted at 5000 g for one hour, then resuspended in approximately 2 mL of chilled DNA extraction buffer. 1 mL of this resuspension was transferred to a 2 mL screw-cap tube with 0.5 g for 0.1 mm zirconium beads, 50 uL 20% SDS, and 700 uL cold equilibrated phenol. This mixture was subjected to bead beating for 2 minutes on a tabletop bead beater (Mini Bead Beater, Biospec Products, Bartlesville, OK), then heated in a 60 °C water bath for 10 minutes before another 2 minutes of bead beating. The mixture was then centrifuged for 10 minutes at 4 °C on a tabletop centrifuge at max speed (~15,000 rpm, Microfuge 20 R, Beckman Coulter, Brea, CA). The aqueous layer was washed 2–4 times with cold equilibrated phenol until the white lipid layer disappeared before precipitation of DNA in a mixture of 0.1 vol 2 M Na acetate and 0.6 vol isopropanol overnight. DNA was pelleted in a tabletop centrifuge for 20 minutes at 4 °C with max speed. The pellet was washed with 70% ethanol, then dried overnight. Pellet was resuspended in 100 μL of elution buffer (Invitrogen, Thermo Fisher Scientific, Waltham, MA).

To amplify the 16S rRNA gene, universal primers flanking the variable 4 (V4) region were used58. For one reaction per sample, 25 ng of template DNA, 5 pmol of each of the forward and reverse primers, and 12.5 μL of 2X HotStart ReadyMix (KAPA Biosystems, Wilmington, MA), and water to a total volume of 25 μL were used. Cycling conditions were as follows: initial denaturation of 95 °C for 3 minutes, 25 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, and a final extension at 72 °C for 5 minutes. Size exclusion was performed by gel electrophoresis on a 1.0% low-melt agarose gel (National Diagnositcs, Atlanta, GA), followed by gel extraction of amplified DNA using a ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Extracted DNA was quantified via a 96-well protocol using manufacturer’s instructions with the Quant-iT™ dsDNA High-Sensitivity Assay Kit, using reagents from a Qubit® dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). Reactions were read on a Synergy 2 Multi-Mode Reader (BioTek, Winooski, VT) following a programmed 3 second shaking period and a 2 minute incubation at 22 °C. Amplified DNA was equimolar pooled, combined with 10% PhiX control DNA, and sequenced using the MiSeq. 2 × 250 v2 kit (Illumina, San Diego, CA) with custom sequencing primers as described by Kozich et al.58.

The program mothur v.1.38.1 was used for further sequence processing38, following a protocol developed from Kozich et al.58, as described in Weimer et al.59. In short, paired-end sequences were assembled into continuous segments and poor-quality sequences were removed. The SILVA 16S rRNA gene reference alignment database v12860 was used to screen for alignment to the v4 region. Preclustering was performed (diffs = 2) to reduce error and computational load, and chimeric sequences were removed (UCHIME61). The 2013 release of the GreenGenes database62 was used to classify sequences with a bootstrap value cutoff of 80. Sequences classifying to cyanobacteria, mitochondria, Eukarya, or Archaea were removed. Bacterial sequences were grouped into operational taxonomic units (OTUs) with 97% sequence similarity. Good’s coverage63 was calculated in mothur. OTU counts were normalized to 10,000 sequences per sample, and normalized OTU counts were used for further analysis. Stacked bar plots were generated in R version 3.4.3 using the package phyloseq v1.22.364.

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