Eznatm soil dna kit

Total bacterial DNA was extracted from the intestinal digesta using the EZNATM Soil DNA kit (D5625-02, Omega Bio-Tek Inc., Norcross, GA, USA) according to the instructions of the manufacturer. The V3-V4 hypervariable regions of the bacterial 16S rDNA were amplified by a two-step PCR method using primers 338F (5′-ACTCCTRCGGGAGGCAGCAG-3′) and 806R (5′-GGACTACCVGGGTATCTAAT-3′) with unique 8-bp barcodes to facilitate multiplexing, and sequencing was carried out with an Illumina sequencing platform using Miseq PE300.

Total bacterial DNA was extracted from the intestinal chyme and mucosa using the EZNATM Soil DNA kit (D5625-02, Omega Bio- Tek Inc., Norcross, GA, USA) according to the instructions of the manufacturer. The V3-V4 hypervariable regions of the bacterial 16S rDNA were amplified by a two-step PCR method using primers 338F (5′-ACTCCTRCGGGAGGCAGCAG-3′) and 806R (5′-GGACTACCVGGGTATCTAAT-3′) with unique 8-bp barcodes to facilitate multiplexing, and sequencing was carried out with an Illumina sequencing platform using Miseq PE300 (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0 (Chen et al., 2018 (link)), and merged by FLASH version 1.2.7 (Magoc and Salzberg, 2011 (link)). Operational taxonomic units (OTUs) with 97% similarity cutoff (Edgar, 2013 (link)) were clustered using UPARSE version 7.1 [3], and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 (Wang et al., 2007 (link)) against the 16S rRNA database using a confidence threshold of 70% (PRJNA889391).

Genomic DNA was extracted from the jejunum mucosa using the EZNA^TM Soil DNA Kit (D5625-02, Omega Bio-Tek Inc., Norcross, GA, USA), as directed by the manufacturer. The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified by a two-step PCR using specific primers (338F, 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R, 5′-GGACTACH VGGGTWTCTAAT-3′) with unique 8-bp barcodes to facilitate multiplexing. The amplicons were sequenced using the Illumina HiSeq sequencing platform, as previously described. The Majorbio Cloud Platform (www.majorbio.com) was used to analyze the raw data. The raw reads were deposited to the Sequence Read Archive (SRA) database (Accession Number: PRJNA876628) of NCBI. A more detailed methodology was described previously [21 ].

The primer sequences of E. coli and 16S were shown in Table S6. PCR products were ligated to the pMD18-T vector system (Takara, Japan), which was subsequently transformed into E. coli strain DH5α. Plasmid DNA carrying the insert was extracted and used as the template for DNA sequencing. Ten-fold serial dilutions of plasmid pMD18-T from 10^-3 to 10^-8 or 10^-3 to 10^-6 were performed to generate standard curve for absolute quantification. Microbial DNA was extracted from the spleen, liver, and colon using the EZNA ^TM Soil DNA kit (Omega Bio-Tek Inc., GA, USA) according to the manufacturer’s protocol. qPCR was performed to quantify E. coli on an ABI 7300 real-time PCR system (Applied Biosystems, USA). The 20-μL PCR reaction mixture contained 10 μL ChamQ SYBR Color qPCR Master Mix (2×), 0.8 μL of each primer, 0.4 μL ROX Reference Dye 1 (50×), and 2 μL of the template DNA. The cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 5 s, 58°C for 30 s, and 72°C for 1 min.

Production water samples (1 L) were centrifuged at 12,000×g for 15 min at 4 ℃ to collect cell pellet. DNA was isolated from the biomass pellets using the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences, Inc., Union City, CA, USA) following the instructions of the manufacturers. For soil samples, DNA was extracted from 2 g of sediments using E.Z.N.A.^TM Soil DNA kit (D5625-01, Omega Bio-Tek, Inc., Norcross, GA, USA), according to its operating manual.
The 2× Taq PCR Master Mix (TaKaRa Bio, Shiga, Japan) was used to set up PCR reactions in 25 μL volume. Of all the primer sets designed in this study, primer set 6’ (ahyA1377_for: 5′-AGCYTSGGCAAGAARGGMTGC-3′, ahyA1843’_rev: 5′-ATGGTCTTRTAYTTDTY CCASAG-3′) was tested as the best one and then chosen to amplify the corresponding nucleotide sequences of this functional gene. PCR reaction conditions were: 98 ℃ for 1 min followed by 32 cycles of 98 ℃ for 15 s, 50 ℃ for 30 s, 72 ℃ for 40 s and a final extension step at 72 ℃ for 10 min. Subsequently, PCR products were pooled and purified with a DNA purification kit (Axygen Biosciences, Inc., Union City, CA, USA).