title: 16S rRNA gene amplicon sequencing of raw milk from Holstein cow which experienced K. pneumoniae clinical mastitis
design: The V4 region of the 16S rRNA gene was amplified via PCR using F548 and R806 primer pair and HotStarTaq Plus (QIAGEN, Germany). PCR reaction included a denaturation stage (95C, 5 minutes), 35 cycles of amplification (95, 30 seconds; 50, 30 seconds; 72, 1 minute), and an extension stage (72C, 10 minutes), followed by holding samples at 4C. Amplicons were purified using Agencourt AMPure XP (Beckman Coulter Inc., USA), then quantified using Quant-iT dsDNA Assay Kit (Invitrogen Inc., USA), as per the manufacturers protocol. Amplicons with sufficient concentration were normalized to a final concentration of 1.5ng/L using DNA/RNA-free water, then 2L of each normalized sample was pooled together prior to sequencing. The pooled library was sequenced using the MiSeq and a 500 cycle V2 MiSeq reagent kit (Illumina Inc., USA).