RNA-seq of ovine abomasal tissue
Total RNA was extracted from the abomasal biopsies as follows: the 645 abomasal biopsy samples were homogenised in RLT buffer (Qiagen Ltd, UK) using a Precellys bead basher (Bertin Instruments, UK) with CK28 bead tubes (Stretton Scientific, UK). Samples were centrifuged at 14,000 g at 4C for 10 mins and the supernatant collected for processing using a RNeasy mini-isolation kit (Qiagen Ltd, UK) according to the manufacturers protocol, including an on-column DNase digestion. RNA quality and integrity were assessed using a Nanodrop spectrophotometer (Thermo Fisher, UK) and a Bioanalyser RNA Nanochip (Agilent Technologies Ltd, UK). The yield of total RNA was determined on a Qubit Fluorometer (Thermo Fisher, UK) using the Broad Range RNA kit (Thermo Fisher, UK). The RNA isolated from three biopsies per animal for each time-point was pooled 1:1:1 by weight to generate the final samples for RNA-seq assessment.