Qiaquick gel extraction kit

Total intracellular viral RNA was extracted from infected cells using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. RT-PCR was carried out using AccuScript (Agilent Technologies), with specific oligonucleotide primers (S1 Table) The amplicons covered the following genomic regions: A1, spanning genomic residues 7626 to 7962; A2, residues 7941 to 8257; and A3, residues 8229 to 8653. Negative controls without template RNA were included in parallel to ascertain the absence of cross-contamination by template nucleic acids. PCR products were purified (QIAquick Gel Extraction kit), quantified (Pico Green assay), and analyzed for quality (Bioanalyzer) prior to Illumina MiSeq sequencing.

After in vitro fermentation of 48 h, bacterium cells of SFSP and CON groups were separated by centrifugation at 8000 g for 10 min from fermentation broth. The total bacterial DNA in each group was extracted by the Power Fecal DNA Isolation Kit (MO BIO, Carlsbad, USA). Agarose gel electrophoresis method was used to evaluate the DNA quality. The V3 regions of 16S rRNA was amplified with universal primers 515F and 806R by PCR assay. The QIAquick Gel Extraction Kit was used for purification of PCR products and then sequenced by the Illumina HiSeq 2,500 platform. Bioinformatics classification of species in the different levels were based on the operational taxonomic units (OTUS) and the sequences with similarity of ≥97% were classified into an OTU unit individually performed by RDP classifier Bayesian algorithm at Usearch (version 7.0 http://drive5.com/uparse/). The biological classification information of each operational taxonomic unit (OUT) unit was obtained by matching the Silva database (http://www.arb-silva.de).