Bacterial DNA amplicon sequencing of bovine rumen
On day 22nd of each experimental period, rumen content (mass and liquid) of each cow was sampled for archaea and bacteria DNA extraction as well for counting total and differential  numbers of protozoa. For rumen archaea and bacteria community sequencing, samples were  collected at 3 hours after morning feeding and for protozoa at zero (0), 3, 6, 9 and 12 hours after  morning feeding. Samples of 50 mL of whole rumen contents were collected on day 22 of each period  through the ruminal cannula at 3 hours after the morning feeding and stored at 80C. After  thawing, samples were separated by phase (liquid and solid). Proportionate amounts of liquid  and solid phase rumen content, as determined by when the rumen was completely emptied and  phases separated and measured, were combined and then processed to isolate DNA following  the procedure detailed in Weimer et al. (2017). The resuspended pellets were then processed to isolate DNA following the bead-beating method described by Weimer et al. (2017). The DNA was resuspended in 10 mM Tris HCl with  1 mM EDTA (pH 8.0), and was quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States), and stored at 4 C before preparation of the DNA library. A two-step PCR was employed to amplify the V3 and V4 regions of the 16S ribosomal RNA gene for bacteria and the V6 through V8 regions of the16S rRNA gene for archaea (KITTELMANN et al., 2013).   PCR clean-up, and sequencing were performed as described (CUNHA et al., 2017). The PCR reactions contained 2550 ng of DNA, 10 M of each primer, 12.5 L of 2X  KAPA HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, United States), and water to  a total volume of 25 L. Cycling conditions were as follows: initial denaturation of 95C for 3  min, 25 cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s, and a final extension at 72C  for 5 min. Gel electrophoresis was performed using a 1.0% low-melt agarose gel (National  Diagnostics, Atlanta, GA, United States), where bands present at 380 bp indicated successful  amplification. Bands were excised from the gel and DNA was extracted from the bands using a ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, United States). No-template negative controls were included for each set of PCRs, and absence of a band in the gel indicated no contamination was present. Extracted DNA was quantified in duplicate on 96-well microplates according to manufacturers instructions for the Quant-iT dsDNA Broad-Range Assay Kit, using reagents from a Qubit dsDNA Assay Kit (Thermo Fisher Scientific, Waltham,  MA, United States), read on a Synergy 2 Multi-Mode Reader (BioTek, Winooski, VT, United  States) after a programmed 3-s shaking period and a 2-min incubation at 22C. The extracted DNA was equimolar pooled. The final library was sequenced using a MiSeq v2 2  250 kit (Illumina, San Diego, CA, United States), with a final library concentration of 10 pmol/L and 10% PhiX control. Custom sequencing primers as described by Kozich et al. (2013) were used.