RNA-Seq of bovine rumen: 0 hour
Three mature, rumen-cannulated, non-lactating Holstein x Friesian cows were used for this experiment.  Stitched nylon bags (10 cm  20 cm) of 100 m2 pore size were filled with 15 g (fresh weight) of the perennial ryegrass Lolium perenne cv. AberDart; PRG. Bags were placed simultaneously in the rumen of each cow shortly after animals were offered the first meal of the morning and removed after 0, 1, 2, 4, and 6 hours.  At each time interval, twelve bags (four from each cow) were withdrawn and the residual plant material in six of the bags (two for each cow) was processed by washing with distilled water (500 ml added to plant material within bags and bags gently squeezed thereafter) to remove loosely attached microbes, followed by oven drying for one replicate sample from each cow and calculation of plant degradation (% dry matter lost).  The remaining six bags (two for each cow) were immediately frozen in dry ice and then stored at -80C prior to RNA extraction. Frozen samples were ground to a fine powder under liquid nitrogen before RNA was extracted using a hot phenol method (Ougham and Davies, 1990).  Library sequencing was completed using an illumina HiSeq 2500 (illumina, California, USA) (100 bp paired end sequencing).