The effect of different concentrations of ruminal VFAs on the circadian rhythm of gene expression in ovine ruminal epithelial cells using RNA-seq
This experiment aimed to investigate the effect of different concentrations of ruminal VFAs on the circadian rhythm of gene expression in ORECs of Grain-diet and Hay-diet groups. The ORECs treated with  Grain-VFAs and Hay-VFAs were collected at six different timepoints (T0, T4, T8, T12, T16, and T20) for the circadian rhythm analysis using RNA sequencing. Overall design: In Grain-diet group, 71.1 mM acetate (AA, prepared with sodium acetate, 241245, Sigma-Aldrich, St. Louis, MO), 20.1 mM propionate (PA, prepared with sodium propionate, P1880, Sigma-Aldrich), 16.0 mM butyrate (BA, prepared with sodium butyrate, 303410, Sigma-Aldrich), 1.7 mM valerate (VA, 75054, Sigma-Aldrich), 1.1 mM isobutyrate (IBA, I1754, Sigma-Aldrich), and 5.3 mM isovalerate (IVA, 129542, Sigma-Aldrich) were added into the culture medium (Grain-VFAs culture medium, GVCM); in Hay-diet group, 60.8 mM AA, 14.6 mM PA, 9.2 mM BA, 1.2 mM VA, 1.0 mM IBA, and 3.0 mM IVA were added into the culture medium (Hay-VFAs culture medium, HVCM) . The total VFAs concentration in the GVCM was 115.78 mM, while the total VFAs concentration in the HCM was 89.79 mM. After preparation, the pH value of the culture medium was adjusted to 7.2 using 7.5% NaHCO3 solution (C0220, Beyotime). Cells were cultured in 6-well plates, when the cells were reached at 70% density, the culture medium was changed to DMEM/F12 containing 100 nM dexamethasone (DEX) and 1% penicillin-streptomycin solution without FBS (Synchronous culture medium, SCM) . The cells were synchronized to the initial circadian rhythm state after incubation with SCM for 2 hours, and then the cells were treatment with GVCM and HVCM for 24 hours. Cells in the Grain-diet and Hay-diet groups were then harvest at 4-hour interval at 24h (T0), 28h (T4), 32h (T8), 36h (T12), 40h (T16), and 44h (T20), each timepoint had 3 replicates per treatment.