16S_MiSeq_of_in_vitro_fermentation_S12_Water (Digesta Control)_10h
At sampling time, a 1 mL aliquot of fermenta was immediately centrifuged at 13,000 x g for 5 min at 4C, and the pellet stored at -80C. DNA was extracted from the pellet using the PowerSoil DNA Isolation kit (MO Bio Laboratories, California, USA), according to the manufacturers instructions with the following modifications. The pellets were dispersed in the PowerBead tubes, to which 60 L of solution C1 was added. This was followed by mechanical shaking using a FastPrep-24 5G (MP Biomedicals, Ohio, USA), with 4 cycles of 5.5 m/s for 60 s, with the tubes being kept in ice for 5 min between each cycle. The tubes were then frozen at -80C and all further steps carried out as per manufacturers instructions within 48 h. DNA was submitted to Massey Genome Service (Palmerston North, New Zealand) for preparation of amplicon libraries for the 16S rRNA gene V3-V4 regions using the 341F and the 806R primers that also included the appropriate Illumina adaptors, index and pad sequences and linkers for downstream sequencing. The purified amplicons were then pooled in equal molarity and the sequencing performed using 2 x 250 base paired-end runs using the Illumina MiSeq v. 2.0 platform.