Powerfecal dna isolation kit

From the scrapped adherent-mucosa, bacterial DNA extracts were obtained by using the PowerFecal DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol. Specific regions of the bacterial 16S rDNA gene were amplified using real-time qPCR. Eubacteria were quantified by real-time qPCR using specific primers (HAD-1: 5′-TGGCTCAGGACGAACGCTGGCGGC-3′ and HAD-2: 5′-CCTACTGCTGCCTCCCGTAGGAGT-3′), annealing at 59 °C for total bacteria. In addition, the V3-V4 region amplification of the 16S rDNA gene was performed and sent to the GenoToul platform (Castanet-Tolosan, France) for MiSeq Illumina sequencing using the MiSeq kit V2 2 × 250 bp. Data quality check and analysis were performed using the INRA-Migale server, as previously published [21 (link)]. In parallel, sulphate-reducing bacteria group were quantified by real-time qPCR using specific primers targeting Dissimilatory Sulfite Reductase A (DSR-1F: 5′-ACSCACTGGAAGCACGGCGG-3′ and DSR-R: 5′-GTGGMRCCGTGCAKRTTGG-3′), Fast SYBR Green MasterMix (Applied Biosystems, Fisher Scientific, Illkirch, France) and StepOne Real-Time PCR system (Applied Biosystems, Fisher Scientific, Illkirch, France).

Genomic DNA (gDNA) was extracted from duodenal fluid using PowerFecal DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) as described in the protocol provided by the manufacturer.

PowerFecal™ DNA Isolation Kit (MoBio Laboratories, Inc) was used for DNA extraction from the intestinal content samples. Amplification and sequencing of the V3-V4 region of the bacterial 16S rDNA gene was performed using barcoded fusion primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC). PCR amplification was then performed under the following conditions: initial denaturation at 98 °C for 30 s, 25 cycles at 98 °C for 10 s, 53 °C for 30 s and 72 °C for 30 s, and final extension at 72 °C for 7 min. The amplicons were pooled in equimolar concentration and sequenced with an Illumina MiSeq platform.

Frozen intestinal samples were homogenized to a powder using individual sterilized mortar and pestles partially submerged in liquid nitrogen. The PowerFecal^® DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, United States) was then used to isolate DNA following the manufacturer’s recommendations. DNA was checked for quality and concentration using a NanoDrop 2000c (ThermoFisher, Wilmington, DE, United States) and gel electrophoresis. DNA samples were then submitted to the Roy J. Carver Biotechnology Center (University of Illinois, Champaign, IL, United States) for preparation and sequencing of 16S rRNA V4 gene amplicons. Sequencing libraries were prepared using 505f and 806r target-specific PCR primers (Caporaso et al., 2011 (link)), CS1 and CS2 spacer pads, sample-specific 10 nucleotide (nt) barcodes, and sequencing adapters. Libraries were then sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, United States). Additionally, microbiota data for one diet-associated and two water-associated samples were obtained from an overlapping study (Bledsoe et al., 2016 (link)), corresponding to the diet and the water supplied to rearing tanks at the time of sampling.

The PowerFecal DNA isolation kit (MO BIO Laboratories, Inc., San Diego, CA) was used to extract DNA per protocols established by the Human Microbiome Project.⁴⁷ Library construction and sequencing was carried out at the New York Genome center using the TruSeq Nano DNA LT Library Preparation Kit (Illumina, Inc., San Diego, CA), which generates 450 bp libraries and sequenced on the HiSeq 2500 System at 2 × 125 bp read length resulting in ~250 M reads.

The feces and intestinal contents were collected under sterile conditions. In brief, on the day of sacrifice, the fresh feces were collected at the time of defecation by scooping the feces into a sterile 15 ml centrifuge tube. For collection of intestinal contents, the intestine was cut into two fragments at the ileocecal junction. The contents were eluted from both fragments of the intestine with 10 ml of sterile normal saline and collected in 15 ml tubes. The tubes were centrifuged at 4,000 rpm for 10 min, and the supernatant was discarded. The feces and intestinal contents from the same mice were mixed and frozen in liquid nitrogen for 15 s and stored at −80°C in the laboratory before shipment.
Samples were packaged with 15 kg dry ice and sent to a company (Novogene Bioinformatics Technology Co., Ltd. in Tianjin, China) where the samples were stored at −80°C until DNA extraction. Total bacterial DNA was extracted from approximately 400–600 mg of each sample using a PowerFecal™ DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions, and was stored at −80°C before further analysis. The DNA concentration and purity was checked on 1% agarose gel, and the DNA was diluted to a 1 ng/μl working stock.

Whole microbial genome DNA was extracted from fecal samples using PowerFecal DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA). Aliquots of extracted genomic DNA were quantified using Qubit dsDNA HS Assay Kit (Life Technologies, Waltham, MA). The DNA was amplified with Illumina adapter and indexed polymerase chain reaction primers. Bacterial 16S ribosomal RNA was sequenced using the Illumina MiSeq sequencing platform (Illumina, Inc., San Diego, CA) as previously described (14 (link)). Further details are given in the supplemental methods (Supplemental Digital Content 1, http://links.lww.com/CCM/E693).