16S sequencing of rumen fluid
Microbial DNA was isolated using QIAmp DNA Stool Kit (Qiagen). Genomic DNA was used to PCR-amplify V4 hypervariable region of 16S rRNA gene using 5Prime HotMasterMix with 515-For-primer (GTGBCAGCMGCCGCGGTAA) and 806-Rev-primer (GGACTACHVGGGTWTCTAAT) described by Kozich et al., 2013 and Hugerth et al., 2014. The PCR condition: 95C for 2 min, 35 cycles of (95C for 30 s, 50C for 60 s and 72C for 90 s), 10 min at 72C. Duplicates of PCR products were pooled and purified using SequalPrep Normalisation Plate (Thermo Fisher Scientific). The DNA libraries were parallel sequenced for 2 x 250bp paired-end reads using the HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 on the rapid-run mode of the HiSeq 2500 system (Illumina). Sequence data were de-multiplexed and converted into FASTQ files using the bcl2fastq2 conversion software, v2.19 (Illumina).