Omega e z n a stool dna kit

Microbial DNA was extracted from each of all the microbial samples (n = 123) from goat kids and mothers using the OMEGA E.Z.N.A. Stool DNA Kit following the manufacturer’s protocol (Omega Bio-Tek Inc., Norcross, GA, United States). DNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States), and the integrity was checked by 1% agarose gel electrophoresis. Concentrations of volatile fatty acids (VFAs) were determined using an Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, United States) following a standard procedure described in previous work (Li et al., 2014 (link)).

The total DNA (30 cecal content samples with low cecal TMA levels and 30 cecal content samples with high cecal TMA levels) was extracted using an OMEGA E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer’s instructions. The V4 region of 16S rRNA was amplified using 515F-806R primers with barcodes. The PCR was carried out using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s recommendations. Library quality was assessed on a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The library was sequenced on an Illumina HiSeq2500 platform to generate 250 bp paired-end reads.

Soil microbiome DNA was extracted by the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., CA) [Omega E.Z.N.A. Stool DNA Kit (Omega Bio-Tek, Inc., USA)]. The extracted DNA was assayed for DNA quality and concentration using a Nanodrop 2000 (ThermoFisher Scientific, Inc., USA). The samples were stored at −20°C for subsequent experiments.