Powermag microbiome rna dna isolation kit

Bacterial DNA was isolated from intestinal content and fecal samples using a PowerMag® Microbiome RNA/DNA Isolation Kit (MO BIO Laboratories) on an epMotion® robot (Eppendorf). DNA sequencing of the V4 region of the bacterial 16S rRNA gene was performed on a MiSeq instrument (Illumina) in paired-end, ∼250 bp format at the University of Michigan Microbial Systems Molecular Biology Laboratories as previously described (Seekatz et al., 2015 (link)).

Bacterial DNA was extracted from samples with the PowerMag Microbiome DNA/RNA Isolation Kit (Qiagen, Hilden, Germany) and the epMotion workstation (Eppendorf, Hamburg, Germany) following manufacturer’s instructions. DNA concentration was assessed with the Quant-IT PicoGreen dsDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). All samples were processed through the MiSeq Wet Lab SOP to prepare the hypervariable V4 region of 16S rRNA gene sequences for Illumina MiSeq sequencing [26 (link), 27 ]. Mock community and negative extraction controls were also included for sequencing [28 (link)]. Once samples were processed, pooled, and normalized to at least 1 nM, they were submitted to the NADC Genomics Facility in Ames, IA for preparation of 250 bp paired-end library and sequencing on the MiSeq instrument (Illumina, Inc. San Diego, CA, USA) using version 2 chemistry.

Bacterial DNA from nasal samples was extracted using the PowerMag Microbiome DNA/RNA Isolation Kit (Qiagen, Hilden, Germany) and the epMotion 5075 workstation (Eppendorf, Hamburg, Germany), following the manufacturer's instructions. DNA concentration was assessed with the Quant-IT PicoGreen dsDNA Kit (Thermo Fisher Scientific, Waltham, MA). All samples were processed through the MiSeq Wet Lab SOP to prepare the hypervariable V4 region of 16S rRNA gene sequences for Illumina MiSeq sequencing (Kozich et al., 2013 (link)): https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/MiSeq_WetLab_SOP.md. Mock community and negative extraction controls were also included for sequencing (Allen et al., 2016 (link)). Once samples were processed, pooled, and normalized to at least 1 nM, they were submitted to the NADC Genomics Facility in Ames, IA for preparation of 250 bp paired-end library and sequencing on the MiSeq instrument (Illumina, Inc. San Diego, CA) using version 2 chemistry.

Bacterial DNA from nasal samples was extracted using the PowerMag Microbiome DNA/RNA Isolation Kit (Qiagen, Hilden, Germany) and the epMotion 5075 workstation (Eppendorf, Hamburg, Germany), following the manufacturer's instructions. DNA concentration was assessed with the Quant-IT PicoGreen dsDNA Kit (Thermo Fisher Scientific, Waltham, MA). All samples were processed through the MiSeq Wet Lab SOP to prepare the hypervariable V4 region of 16S rRNA gene sequences for Illumina MiSeq sequencing (Kozich et al., 2013 (link)): https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/MiSeq_WetLab_SOP.md. Mock community and negative extraction controls were also included for sequencing (Allen et al., 2016 (link)). Once samples were processed, pooled, and normalized to at least 1 nM, they were submitted to the NADC Genomics Facility in Ames, IA for preparation of 250 bp paired-end library and sequencing on the MiSeq instrument (Illumina, Inc. San Diego, CA) using version 2 chemistry.