Dneasy powersoil dna extraction kit

DNA was extracted from the stored cotton swab tips using a DNeasy Power Soil DNA Extraction Kit (Qiagen, Venlo, Netherlands). Cotton swab tips were removed with sterile scissors and then added directly to the kit beaded tube, after which the standard kit protocol from the manufacturer was followed. DNA was extracted from the dissected adult digestive tracts using the same DNeasy Power Soil DNA Extraction Kit. The manufacturer’s protocol was followed with the addition of a preliminary 10-minute heating step at 75°C prior to beaded tube vortexing, which assisted in degradation of the intestinal tissue. DNA from anal secretion samples was extracted using a DNeasy Blood and Tissue DNA Extraction Kit (Qiagen, Venlo, Netherlands) following the standard kit protocol. Pure DNA extracts were sent to the University of Colorado, Boulder for high throughput Illumina sequencing on the MiSeq platform following previously described methods [30 ].

Oral and stool samples were thawed on ice prior to DNA extraction. DNA was extracted using the DNeasy Powersoil DNA Extraction kit (Qiagen Inc, Hilden, Germany) to generate high molecular weight DNA free of PCR inhibitors. Samples were examined for DNA integrity by agarose gel electrophoresis and Nanodrop (ThermoFisher Scientific, Waltham MA). DNA was quantified using SYBR Gold (ThermoFisher) prior to downstream applications.

Seawater was decanted from the self-clumping BBD mats and mats were either frozen immediately (Ft. Lauderdale sample) or preserved (Belize and Looe Key samples) with 5–10 volumes of RNAlater (Qiagen, Germantown, MD, USA) before freezing at −80 °C. DNA and RNA were co-extracted with an Allprep DNA/RNA mini kit (Qiagen, Germantown, MD, USA) from the Belize samples. RNA was treated with DNase I (New England Biolabs, Ipswich, MA, USA), concentrated with RNA Clean & Concentrate (Zymo Research, Irvine, CA, USA), and cDNA was synthesized with a VILO Superscript cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). DNA was extracted from the two Florida field samples and from the cyanobacterial enrichment culture with a Dneasy Powersoil DNA extraction kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions with the addition of 12 μL of proteinase K (New England Biolabs, Ipswich, MA, USA) to the bead tube with solution C1 and incubated 30 min at room temperature before the bead beating step.

Total DNA was extracted from ∼0.25 g of soil using a DNeasy PowerSoil DNA extraction kit (Qiagen, Hilden, Germany). Extracted DNA was assessed using a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit (Thermo Fisher Scientific, Waltham, MA, USA). Unamplified TruSeq libraries were prepared for 4 DNA samples prior to sequencing on an Illumina HiSeq-2000 platform (Illumina, San Diego, CA, USA) at the DOE JGI. Raw Illumina reads were trimmed, quality filtered, and corrected using bfc (version r181) with the following options: -1 -s 10g -k 21 -t 10. Following quality filtering, reads were assembled using SPAdes (v3.11.1) (92 (link)) with the following options: -m 2000 –only-assembler -k 33,55,77,99,127 –meta -t 32. The entire filtered read set was mapped to the final assembly, and coverage information was generated using BBMap (v37.62) (91 ) with default parameters except ambiguous=random. The version of the processing pipeline used was jgi_mga_meta_rqc.py, 2.1.0. Of the 28 metagenome samples sequenced, only 4 were selected for inclusion in analysis for this study because they corresponded to those samples sorted using FACS.

Total DNA was extracted from 2 to 5 g of grounded halite using the DNeasy Powersoil DNA extraction kit (QIAGEN). The DNA was cleaned with 3× AMPure XP Beads (Beckman Coulter) before library construction. The KAPA HyperPlus kit (Roche) was used to construct the genomic libraries with 10 ng of DNA, following the manufacturer’s instructions and with the DNA from each nodule barcoded separately. Final library size selection was made with 0.5× and 0.7× KAPA HyperPure beads (Roche) ratio to recover fragments 300–500 bp in length. Five libraries for Salar Grande and 3 for the other locations were paired-end sequenced by Novogene (https://en.novogene.com/) with 150 bp reads length using the Illumina NovaSeq 6000 platform.

Total genomic DNA was extracted from the pellet using the DNeasy Powersoil DNA extraction kit (QIAGEN), as recommended by the manufacturer. The quantity and quality of the extracted DNA were determined using an IMPLEN NanoPhotometer NP80 - All-in-One Spectrophotometer. All analyses were performed in duplicate.

Each WWTP's influent (raw/untreated wastewater) and effluent (treated wastewater) received a 1-L composite sample. Composite samples made up of many subsamples were taken, for example, one small sample was taken, followed by a 30-second interval, and then the next sample was taken, and so on, until the required sample volume (1 L) was reached. The samples were transported to the lab in an ice-filled cooler box, kept at 4 °C, and analyzed within 48 h. Per WWTP, two samples were taken (influent and effluent (post-chlorination)). Samples were homogenized before analysis, and 50 mL subsamples were taken and centrifuged at 3000 rpm for 20 min, with the supernatant discarded and the pellet used for DNA extraction. The DNA was extracted using a DNeasy Powersoil DNA extraction kit (QIAGEN) according to the manufacturer's instructions, with no changes. IMPLEN NanoPhotometer NP80 – All-in-One Spectrophotometer was used to determine the quantity and quality of the extracted DNA. All of the analyses were carried out in triplicate.