16S rumen fluid
Sequencing libraries were prepared using a two-step PCR workflow. The V4 region of the 16S rRNA gene was amplified using region-specific primers (V4_515F and V4_806R) and the KAPA HiFi HotStart PCR Kit. In the first PCR, 50 ng of genomic DNA was amplified under the following conditions: 3 min at 95 C, 26 cycles of (30 s at 95 C, 30 s at 64 C, 30 s at 72 C), and a final 5 min at 72 C. Amplicons were confirmed via agarose gel electrophoresis. A second PCR was performed to index the amplicons using Illumina barcoded adapters (Nextera XT v2 Index Kit) under similar conditions with 10 cycles. Indexed libraries were normalized, pooled, and quantified using the Qubit dsDNA HS Assay. Sequencing was performed on an Illumina HiSeq X Ten platform with 150  2 cycles, generating at least 0.7 million paired-end reads per sample. Data were demultiplexed using bcl2fastq v2.20, yielding FastQ files.