Masterpuretm dna purification kit

DNA was extracted from samples using MasterPure^TM DNA Purification Kit (Epicentre® Biotechnologies) in accordance with the manufacturer protocol. Freeze-dried samples were weighed prior to DNA extraction. Extracted DNA was then assessed by measuring the absorbance at 260 and 280 nm (Nanodrop®, Thermo Scientific) with 260/280 nm absorbance ratio for all measured samples comprised between 1.8 and 2. DNA concentration was calculated according to tissue weight (ng of DNA/μL/mg of dry tissue).

DNA from oral biofilm and gut samples of six animals per group was extracted using the Master pureTM DNA Purification Kit (Epicentre^® Illumina Company, Madison, WI, USA). A barcoded primer set Bakt_341F CCTACGGGNGGCWGCAG and Bakt_805R GACTACHVGGGTATCTAATCC [25 (link)] was used to amplify the hypervariable V3–V4 region of 16SrRNA. DNA was sequenced by ByMyCell (Ribeirão Preto, São Paulo, Brazil) using the Illumina MiSeq 2 × 250 platform. Data were submitted to Sequence Read Archive (SRA) under BioProject identification #PRJNA994574.

Genomic DNA was extracted from PWBCs using the MasterPureTM DNA purification kit (Epicenter, Madison, WI, USA) and quantified with the Pico Green dsDNA Quantitation Reagent (Invitrogen, Carlsbad, CA, USA). In order to convert cytosine into uracil, high-quality DNA samples (500 ng) were treated with sodium bisulfite using the EZ-96 DNA Methylation Kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s protocol. Illumina Infinium Human Methylation 450k BeadChip (Illumina, San Diego, CA, USA) was employed to measure DNA methylation levels of CpG sites across the human genome. This analysis was conducted in the Unidad de Genotipado y Diagnóstico Genético from the Fundación de Investigación Clínico de Valencia, as detailed elsewhere [64 (link)].

Genomic DNA of VP152 strain was extracted using Masterpure^TM DNA purification kit (Epicenter, Illumina Inc, Madison, WI, USA) and subjected to RNase (Qiagen, USA) treatment (Ser et al., 2015 (link)). The DNA quality was quantified using NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and a Qubit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Illumina sequencing library of genomic DNA was prepared using Nextera^TM DNA Sample Preparation kit (Illumina, San Diego, CA, USA) and library quality was validated by a Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Palo Alto, CA, USA) prior to sequencing. The genome of VP152 strain was sequenced on MiSeq platform with MiSeq Reagent Kit 2 (2 × 250 bp; Illumina Inc, San Diego, CA, USA). The trimmed sequences were de novo assembled with CLC Genomic Workbench version 5.1 (CLC Bio, Denmark).

Cells harvested from 10 g of fermented quinoa flour were resuspended in sterile saline and treated with 2.5 mM propidium monoazide (PMA) stock solution (1.3 mg/mL PMA in 20% DMSO; Biotium, Inc., Hayword, CA, USA) to eliminate dead bacterial and extracellular DNA as described by (Pan and Breidt, 2007). PMA-treated samples were stored as cell pellets at −20 °C until DNA extraction was performed. Total genomic DNA was extracted using a MasterPure^TM DNA Purification Kit (Epicentre, Madison, WI, USA). The 16S rDNA gene regions V3 to V4 were amplified by PCR using the Bakt_341F/Bakt_805R primers [37 (link)]. Primers were barcoded as described by the manufacturer for the Illumina MiSeq sequencing technology. For the fungi analysis, primers ITS1F/ITS2 were used. Sequencing services were obtained from the Carver Biotech Laboratory at the WM Keck Center for Comparative and Functional Genomics (Chicago, IL, USA). Twenty-two samples corresponding to 3 quinoa flour-type fermentations including real, red and black collected at different time points (day 1, 6 and 8) were subjected to DNA extractions and sequencing data processing.

Amplification of bacterial 16S rDNA genes by polymerase chain reaction (PCR) was carried out to identify the bacteria strain according to a published method [20 ]. Genomic DNA was extracted using MasterPureTM DNA Purification Kit (Epicentre Inc., Madison, WI, USA). The PCR amplification and purification processes were conducted as described previously [20 ]. PCR product sequence alignment was done using GenBank BLASTN program, followed by phylogenetic analysis using the Molecular Evolutionary Genetic analysis version 6.0 [21 (link),22 (link)].

DNA quantification was performed on extracted DNA after 4, 7, and 10 days of WJ-MSCs culture in HEMOCOLLAGENE^® foam, using a MasterPure^TM DNA Purification Kit (Epicentre, Biotechnologies, Strasbourg, France) in accordance with the manufacturer’s protocol. The quantity of extracted DNA was assessed by measuring the absorbance at 260 nm (Nanodrop, Thermo Scientific, Villebon-sur-Yvette, France) and the 260/280 nm absorbance ratio for all measured samples was between 1.8 and 2.