Study the role of different concentrations of VFAs on histone modifications in ovine ruminal epithelial cells at am and pm using CUT&Tag-seq

This experiment employed CUT&Tag-seq (Cleavage Under Targets and Tagmentation with sequencing) to explore the mechanism of how different concentrations of VFAs regulate ruminal epithelial histone modifications under the Grain-diet and Hay-diet patterns in both am and pm. Cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were havest for CUT&Tag-seq experiments, n=3 pooled biological replicates per library. The primary histones used for CUT&Tag were Acetyl-Histone H3 (Lys27) Rabbit mAb (H3K27ac, 8173S, CST), Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (H3K9ac, 9649S, CST), and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (H3K4me3, 9751S, CST). 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 HVCM 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). ORECs were cultured with GVCM and HVCM for 24 hours after synchronized to the initial circadian rhythm using SCM contained 100 nM DEX. After 24 hours (T0), cells in the Grain-diet and Hay-diet groups were harvest at 32h (T8) and 44h (T20), which were defined as am and pm, respectively. Finally, cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were rapidly frozen under -80°C conditions for subsequent CUT&Tag-seq experiments (H3K27ac, H3K9ac, and H3K4me3), n=3 pooled biological replicates per library.