title: Target domain Eukaryota amplicon sequencing of bos taurus calf rumen metagenome: animal ear tag
design: Eight bull calves (Friesian  Jersey cross) had access to colostrum for four days post-partum to ensure a good immune status before being fed a standard diet. Methane measurements and rumen content collections were carried out every two weeks until weaning (at 10 weeks of age), and every four weeks after weaning, until week 24. In total, eight methane 24 h measurements were carried out over the 23-week period. Live weights of the animals were determined weekly, and dry matter intake (DMI) was determined on a daily basis until week 10, and then every four weeks during methane measurements in respiration chambers. Rumen fluid sampling was carried out via stomach tubing after the calves were released from the respiration chambers. Rumen samples were subsampled for microbial community analysis via DNA extraction (0.9 ml). Subsamples for microbial community analysis were snap-frozen and stored at -20 C. The samples for DNA extraction were thawed in a 25 C water bath and an aliquot of 200 l of the rumen contents were collected into the pH 8 phenol-chloroform solution (Sigma-Aldrich, St. Louis, MO, USA) using a wide bore tip. The nucleic acids were then extracted following the combined phenol-chloroform and bead-beating method (Rius et al., 2012). DNA was normalized, amplified using barcoded primers targeting variable regions V1-V3 of the bacterial and V6-V8 of the archaeal 16S rRNA gene, or variable regions V5-V8 of the eukaryote 18S rRNA gene and amplicons were pooled and sent to Eurofins Genomics (Ebersberg, Germany) for 454 Titanium pyrosequencing.