Agilent bioanalyzer high sensitivity dna assay

Genomic libraries from the two samples (i.e., animals) were constructed following the manufacturer's recommended protocol with reagents supplied in Illumina's DNA sample preparation kit. Genomic DNA was sheared using a BioRuptor (Diagenode, Denville NJ, USA) to generate fragments. The resulting 3′ and 5′ overhangs were removed by an end repair reaction using a 3′ to 5′ exonuclease activity and polymerase activity to blunt the fragment ends. A single adenosine was added to the 3′ ends of the blunt fragment followed by the ligation of Illumina adapters. The adapter-ligated fragments were then size selected using an agarose gel. Fragments of average length 436 (animal 1) or 477 (animal 2) bp were recovered from the gel slice by elution and ethanol precipitation. Each purified library was quantified with a Qubit assay and fragment size was confirmed by Agilent BioAnalyzer High Sensitivity DNA assay.

For multiplex PCR two customized Ion AmpliSeq primer pools for a total of 1255 amplicons were used (Life Technologies, Darmstadt, Germany). Primer sequences are given in Supplementary Table S5. The DNA input per reaction was 10 ng. Multiplex PCR was performed for 24 target genes (Table 2), most of which are involved in NF-κB signaling. The PCR program consisted of 99°C for 2 minutes, followed by 23 cycles of 99°C for 15 seconds and 60°C for 4 minutes. Libraries were prepared with the NEBNext Ultra DNA Library kit (NEB, Ipswich, UK) for sequencing on the Illumina platform. Purified libraries were quantified with the KAPA library quantification kit (Peqlab, Erlangen, Germany). Library size and quality was determined with the Agilent Bioanalyzer high sensitivity DNA assay (Agilent, Santa Clara, USA). Sequencing was performed on an Illumina MiSeq apparatus with 150 bp paired end reads.

Following DNA extraction, qPCR, and quality checks, samples were submitted to Salient Genomics (Krum, TX) for 16S rRNA gene V4 region sequencing using Illumina MiSeq 2 platform. Samples were prepared and amplified using the Illumina 16S rRNA gene metagenomic library prep guide (Illumina, #15044223 Rev. B). Samples were amplified by PCR and PCR product was cleaned using AmPure XP paramagnetic beads (Beckman Coulter, Indianapolis, IN). A second round of PCR to label sample DNA fragments with indices was done using TruSeq UDI kit (Illumina, San Diego, CA) with 10 base pairs each. Second PCR product was cleaned using AmPure XP paramagnetic beads (Beckman Coulter, Indianapolis, IN). Concentrations were determined for each sample using Qubit HS DNA assay (cat.# Q32851, Invitrogen, Waltham, MA) on Qubit Fluorometer (ThermoFisher, Waltham, MA) and the length of samples was verified using the Agilent Bioanalyzer High Sensitivity DNA assay (Agilent, Santa Clara, CA). Samples were then diluted and run on Illumina MiSeq 2 benchtop platform. Secondary analysis was performed using MiSeq Reporter software and fastq files were generated for downstream analysis.