Enza bacterial dna extraction kit

Colony C2-P2-2 and C2-P2-3 were cultured in 6 mL Gifu media, respectively. Bacterial cells were then pelleted and washed, and bacterial DNA was extracted with ENZA bacterial DNA extraction kit (Omega Biotek, D3350) based on manufacture’s protocol. For genomic preps, DNA was sheared to ~ 500 bp size using a Covaris S2 Focused-ultrasonicator, after which 10 ng of sheared DNA was used to make sequencing libraries with the NEBNext Ultra II DNA Library Prep Kit following manufacturer’s recommended protocol with 9 cycles of PCR. Libraries were size selected using AmpureXP beads to 500–700 bp, loaded onto a flowcell, and sequenced on an Illumina MiSeq sequencer to generate 2 × 300-bp paired-end reads.

Pre-sorted or post-sorted bacteria were subjected to DNA extraction with the ENZA bacterial DNA extraction kit (Omega biotek, D3350) based on manufacture’s protocol. 5–22 ng DNA was used with the NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0 library prep kit (PerkinElmer) following manufacturer’s recommended protocol with between 11 and 20 PCR cycles depending on sample input. PCR amplicons were all pooled and gel purified to select the target amplicon region. Libraries were loaded onto a flowcell and sequenced on an Illumina MiSeq sequencer to generate 2 × 300-bp paired-end reads. We collected 0.2 and 3 million reads per sample for 16S rRNA and whole genome sequencing analyses, respectively. 16S rRNA gene sequencing data was processed with Illumina Basespace 16S metagenomic sequencing application for microbiome taxonomic annotation. The taxonomic classification step used ClassifyReads, a high-performance implementation of the Ribosomal Database Project (RDP) Classifier developed by Wang et al.^{53 (link)}.