Rumen Microbiome Analysis via 16S rRNA Sequencing

The metagenomic DNA of 21 rumen fluid samples was extracted by the cetyltrimethylammonium bromide method. DNA was dissolved in the 200 µL of elution buffer and then preserved at −20 °C. DNA integrity and concentration were determined by 1.5% agarose gel electrophoresis and NanoPhotometer^® spectrophotometer (Implen, Westlake Village, CA, USA), respectively. For rumen bacterial analysis, universal primer pairs (515F-806R) with barcodes were used to PCR amplify the V4 region of the 16S rRNA gene (Li et al., 2016 (link)). PCR amplification was done by using Phusion^® High-Fidelity PCR Master Mix with GC Buffer from New England BioLabs. The amplified products were detected by 2% agarose gel electrophoresis and purified by QIAquick PCR Purification Kit to build libraries. The libraries were built according to the manufacturer’s protocol using TruSeq^® DNA PCR-Free sample preparation kit. The libraries were then quantified by using Qubit dsDNA High Sensitivity Assay kit by Invitrogen. The Illumina HiSeq2500 PE250 system was used for paired-end sequencing using the standard protocol.