Dneasy powerlyzer microbial kit

Identification of strain S14E4C was done based on 16S rRNA gene sequencing: the genomic DNA of strain S14E4C was extracted using a DNA extraction kit (DNeasy Power Lyzer Microbial Kit, Qiagen, Germany). To confirm the identities of the isolates the 16S rRNA gene was amplified (PCR) from the extracted genomic DNA using the universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGCTACCTTGTT ACGACTT-3′) [5 (link)] (LGC Genomics, Berlin, Germany). The 16S rRNA gene sequence of strain S14E4C was compared with references in the EzTaxon database [6 (link)] and the NCBI Nucleotide database using BLAST [7 (link)] to identify closely related bacteria.

Soil isolates were obtained from agarose plugs of gen 1, 2, and 3 by serially diluting the plugs with sterile water and spread plating onto LB agar media (for bacteria) and potato dextrose agar (PDA) media (for fungi), supplemented with mercuric chloride at a concentration of 5 μg/ml of Hg. Isolation of strains was performed as shown before (Jaswal et al., 2019a (link), b (link)). DNA was extracted from the biomass that was colonizing the DC and MT gels as well as the isolated bacterial and fungal strains using DNeasy PowerLyzer Microbial Kit (Qiagen Inc., Germantown, MD, United States). Bacterial and fungal strains were taxonomically identified using the 16S and 18S rDNA sequence analysis. PCR was performed using 27F-1492R universal bacterial primers (Lane, 1991 ), using the following program: initial denaturing at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, followed by a final extension step of 72°C for 5 min. Fungal PCR was performed with FR1/NS1 universal fungal primers (Vainio and Hantula, 2000 (link)), with denaturing at 95°C for 8 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 47°C for 45 s, extension at 72°C for 60 s, and a final extension step of 72°C for 10 min. The sequences obtained were identified for taxonomy using the NCBI BLAST workflow.

Genomic DNA from the methanogen Methanosarcina barkeri DSM 8687 (Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, Germany) in the five fen cores was extracted according to the manufacturer’s instructions in triplicate with a DNeasy® Powerlyzer® Microbial Kit (Qiagen, Hilden, Germany) and quantified with a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Life Technologies, Roskilde, Denmark). Simultaneously, the mcrA (methyl-coenzyme M reductase) gene copy numbers were determined by counting cells (100 × 1.30 plan-neofluar Zeiss oil immersion) in an anaerobic Methanosarcina barkeri culture after cells of known optical density were fixated in 4% formaldehyde solution and stained with 1:10 diluted Acridine Orange. With the M. barkeri DNA extracts as standards, the samples’ mcrA gene abundance was measured with qPCR utilising 10 µM primers mlas (5’-GGTGGTGTMGGDTTCACMCARTA-3’) and mcrA-rev (5’-CGTTCATBGCGTAGTTVGGRTAGT-3’)¹⁶ , 5 × HOT FIREPol® EvaGreen® qPCR Supermix Polymerase (Solis Biodyne, TAG Copenhagen A/S, Copenhagen, Denmark) at 95 °C for 12 min., followed by 35 cycles of 15 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C.

Sediment slurry (5 mL) and liquid (1.8 mL) samples for nucleic acid
extraction were collected from the bioaugmented treatments and the
sediment-free controls, respectively, at each sampling point. DNA
was extracted from slurry samples with the DNeasy PowerSoil Pro Kit
(Qiagen, MD), after centrifugation (20 min; 5000g) and decanting of supernatant. DNA was extracted from sediment-free
samples with the DNeasy Powerlyzer Microbial kit (Qiagen, MD). DNA
concentrations were measured with the Qubit dsDNA high sensitivity
assay kit and the Qubit 4 fluorometer (ThermoFisher Scientific, Waltham,
MA). The bphA gene abundance was measured with qPCR
with an ABI 7000 Sequence Detection System (Applied Biosystems, Grand
Island, NY) as described previously.^{58 (link)} Briefly,
each reaction (20 μL) contained 10 μL of Power SYBR Green
PCR Master Mix (Invitrogen, Carlsbad, CA), 0.3 μM of forward
and reverse bphA primers (Table S2), and 0.1 μL of bovine serum albumin (20 mg/mL; New
England Biolabs, Ipswich, MA). A standard curve using known amounts
of bphA cloned into the 2.1-TOPO vector, prepared
in triplicate was used to quantify bphA abundance.
Melt curve analysis revealed single peaks in both the standards and
samples at a temperature of 86.3 °C. Additional primer and QA/QC
details that satisfy MIQE guidelines^{60 (link)} are
in Tables S2 and S3.

Skin sampling using the wet swab method was performed at baseline and 12 weeks. Samples were collected as described by the Human Microbiome Project^{41 (link)}. A sterile 4 cm square template was placed on the inner arm to mark the sampling area. The collection swab (CatchAll®Sample Collection Swab (Epicenter, Illumina, Madison, WI) was moistened with buffer (50 mM Tris buffer [pH 7.6], 1 mM EDTA [pH 8.0], and 0.5% Tween-20) and the area within the template was swabbed for 30 s rubbing the swab back and forth about 50 times applying firm pressure. The swabs were placed into bead solution for DNA extraction using DNeasy Powerlyzer microbial kit (Qiagen, Valencia, CA) and vortexed for 30 sec. The quality of the extracted DNA was confirmed using the Nanodrop 1000 (Thermo Fisher Scientific, Wilmington, DE).

Fixed dairy product samples were centrifuged at 18,000× g for 20 min and the pellets were used for DNA extraction, which was performed using DNeasy PowerLyzer Microbial Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, including a bead-beating stage using a FastPrep-24™ 5G grinder (MP Bio, Santa Ana, CA, USA). Amplicon libraries of the V4 region of the 16S rRNA gene were prepared as described previously [22 (link)] using a pair of primers 515F [23 (link)] (5′-GTGBCAGCMGCCGCGGTAA-3′)-Pro-mod-805R [24 (link)] (5′-GGACTACHVGGGTWTCTAAT-3′). The libraries were sequenced using a MiSeq system (Illumina, San Diego, CA, USA). The libraries were prepared and sequenced in two replicates for each sample. All sequencing data were deposited into the NCBI SRA database under BioProject number PRJNA789261 (Table S1).

Whole-genome sequencing was performed for the six Sm-resistant strains listed above, as well as the wild-type parent strain. Genomic DNA samples were produced with a final concentration greater than 20 ng/µL, using Proteinase K-mediated lysis followed by column purification using the DNeasy PowerLyzer Microbial Kit (Qiagen). These seven samples were sent to Microbial Genome Sequencing Center (MiGS) for Illumina sequencing to return 200 Mbp of data per strain. Resulting FASTQ files were inputted with the GenBank file for the C58 strain of A. fabrum into the command-line tool, breseq, using Windows Subsystem for Linux and R (46 (link)). The breseq output allowed for the discernment of sequence variants compared to the reference. Whole-genome raw sequence reads for all seven strains are available on NCBI under the BioProject accession number PRJNA993692 (https://www.ncbi.nlm.nih.gov/sra/PRJNA993692).