Dneasy powersoil dna isolation kit

Genomic DNA was extracted from 0.25 g of rhizosphere soil from each sample by using the DNeasy PowerSoil DNA Isolation kit (Qiagen), following the manufacturer protocol. DNA yield and quality were checked both by electrophoresis in 0.8% (w/v) agarose gels stained with GelRed and visualized under UV light, and by using a Qubit 3.0 fluorometer (Life Technologies, Grand Island, NY). DNA from each individual sample was sequenced using the Illumina MiSeq platform at the genomics service of the Institute of Parasitology and Biomedicine “López Neyra” (CSIC), Granada, Spain. Prokaryotic libraries were constructed by amplifying the hyper-variable V3–V4 regions of the 16S rRNA gene using the primer pair 341F (5′-CCTACGGGNBGCASCAG-3′) and 806R (5′-GACTACNVGGGTATCTAATCC-3′) according to Takahashi et al. (2014) (link). These amplicons were tagged to be attached to PNA PCR clamps to reduce plastid and mitochondrial DNA amplification (Lundberg et al., 2013 (link)). Fungal libraries were constructed by amplifying the ITS2 region using the primer pair ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990 (link)) and fITS7 (5′-GTGARTCATCGAATCTTTG-3′) (Ihrmark et al., 2012 (link)). Both runs were sequenced using a paired-end 2x300bp (PE 300) strategy.

The total DNA was extracted from 1 g of sludge sample using a DNeasy PowerSoil DNA isolation kit (Qiagen, Hilden, Germany). A total of about 1.5 μg of DNA was obtained. A metagenomic DNA sample was sequenced using the Illumina (Illumina, San Diego, CA, USA) platform. The TruSeq DNA library was prepared using NEBNext^® Ultra™ II DNA Library Prep Kit (New England Biolabs, Hitchin, UK) and sequenced on the Illumina HiSeq2500 instrument (250 nt single-end reads). Adapter sequences and low-quality reads were removed using Cutadapt [24 (link)] and Sickle (https://github.com/najoshi/sickle accessed on 10 November 2020), respectively. A total of about 50 million high-quality reads were obtained. The reads were de novo assembled into contigs using MetaSPAdes v. 3.7.1 [25 (link)]. The contigs were binned into MAGs using MetaBAT v. 2.12.1 [26 (link)]. Completeness of the MAGs and their possible contamination were estimated using the CheckM v. 1.0.13 tool [27 (link)]. The taxonomic assignment of the obtained MAGs was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk) v. 1.1.1 [28 (link)] and Genome Taxonomy Database (GTDB) [29 (link)].
Gene search and annotation were performed using the RAST server 2.0 [30 (link)] followed by manual correction by the searches of predicted protein sequences against the National Center for Biotechnology Information (NCBI) databases.

The 3 points sampled in each CT with the triplicates were pooled (3L × 3) to testify on the total of autochthonous diversity and then filtered using a sterile mixed nitrocellulose filter with a porosity of 0.22 µm (MF-Millipore GSWP04700, Billerica, MA, USA). Total DNA for each climate was extracted using a DNeasy^® PowerSoil^® DNA isolation kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Mechanical disruption of the cells was performed by shaking at 30 Hz for 2 min using a Retsch Mixer Mill MM400 (Retsch, Haan, Germany).

Fecal samples were collected from female and male 8-week-old WT and Batf3^-/- mice, snap-frozen, and stored at -80°C. DNA was isolated using the DNeasy PowerSoil DNA Isolation Kit (Qiagen) following the manufacturer’s protocol. The V4 region of the 16S gene was amplified and barcoded using 515f/806r primers and 250x2 bp sequencing was performed on an Illumina MiSeq system. Raw data were processed using DADA2 scripts in R platform and quality-filtered reads (~50,862 reads per sample) were used to identify amplicon sequence variants (ASV) by closed reference picking against the Silva database (54 (link)). Abundance of selected bacterial strains were confirmed by qPCR using 25 ng of fecal bacterial DNA and specific 16S rRNA primers for Mucispirillum schaedleri (ASF457), Akkermansia muciniphila, Bifidobacterium, Bacteroides sp. (ASF519) (primer sequences are listed in Supplemental Table). qPCRs were performed in duplicate using SsoAdvanced Universal SYBR® Green Supermix. Relative abundance for each strain was quantified by normalizing the quantity of each specific 16S rRNA gene to the total amount of 16S bacterial DNA. 16S rRNA sequences generated in this study are publicly available. This data can be found here: ENA, accession number PRJEB50182 (https://www.ebi.ac.uk/ena/browser/home).

DNA was extracted from the collected sample using a DNeasy^® PowerSoil DNA Isolation Kit (QIAGEN, Hilden, Germany), and the V1-V2 region of the 16S DNA was amplified using a MiSeq^® instrument (Illumina Inc., San Diego, CA, USA) with a universal primer targeting 16S rRNA (27F mod: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNN- AGRGTTTGATYMTGGCTCAG), (338R: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN-TGCTGCCTCCCGTAGGAGT) and sequenced according to the manufacturer’s instructions. The sequences were clustered into operational taxonomic units based on a 97% homology cutoff. We also analyzed functional predictors. The sequences were analyzed using the QIIME pipeline. The obtained sequences were systematically compared to existing sequences using the EzBioCloud 16S database. Moreover, functional predictive analyses were performed by creating a functional composition table using PICRUSt (ver.1.1.1) based on the Kyoto Encyclopedia of Genes and Genomes ortholog.

Extraction of DNA from water and sediment fractions of each sample was performed using a DNeasy PowerWater DNA isolation kit or DNeasy PowerSoil DNA isolation kit (Qiagen), respectively, per manufacturers’ instructions. The extracted DNA was then eluted with 100 μl of 10 mM Tris elution buffer provided by the manufacturer. Concentration and purity of DNA in each sample was assessed using a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, Madison, WI, United States). All sample of extracted DNA were stored at −80°C until PCR amplification.