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). 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 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.

本实验采用CUT&Tag测序（CUT&Tag-seq，Cleavage Under Targets and Tagmentation with sequencing），探究不同浓度挥发性脂肪酸（Volatile Fatty Acids，VFAs）在谷物日粮与干草日粮模式下，于上午与下午时段对瘤胃上皮细胞组蛋白修饰的调控机制。采集谷物-上午（Grain-am）、谷物-下午（Grain-pm）、干草-上午（Hay-am）、干草-下午（Hay-pm）四个处理组的细胞用于CUT&Tag测序实验，每个文库设置3个混合生物学重复样本。本次CUT&Tag实验所用的核心抗体包括：乙酰化组蛋白H3（Lys27）兔单克隆抗体（H3K27ac, 8173S, CST）、乙酰化组蛋白H3（Lys9）兔单克隆抗体（C5B11, H3K9ac, 9649S, CST）以及三甲基化组蛋白H3（Lys4）兔单克隆抗体（C42D8, H3K4me3, 9751S, CST）。

整体实验设计如下：谷物日粮组培养基（Grain-VFAs培养基，GVCM）中添加：71.1 mM乙酸盐（AA，以乙酸钠配制，货号241245，Sigma-Aldrich，美国密苏里州圣路易斯市）、20.1 mM丙酸盐（PA，以丙酸钠配制，货号P1880，Sigma-Aldrich）、16.0 mM丁酸盐（BA，以丁酸钠配制，货号303410，Sigma-Aldrich）、1.7 mM戊酸盐（VA，货号75054，Sigma-Aldrich）、1.1 mM异丁酸盐（IBA，货号I1754，Sigma-Aldrich）以及5.3 mM异戊酸盐（IVA，货号129542，Sigma-Aldrich）；干草日粮组培养基（Hay-VFAs培养基，HVCM）中添加：60.8 mM乙酸盐（AA）、14.6 mM丙酸盐（PA）、9.2 mM丁酸盐（BA）、1.2 mM戊酸盐（VA）、1.0 mM异丁酸盐（IBA）以及3.0 mM异戊酸盐（IVA）。GVCM中挥发性脂肪酸总浓度为115.78 mM，HVCM中挥发性脂肪酸总浓度为89.79 mM。培养基配制完成后，采用7.5%碳酸氢钠溶液（货号C0220，碧云天生物技术（Beyotime））将培养基pH值调至7.2。

细胞培养流程如下：将瘤胃上皮细胞（ORECs）接种于6孔板中培养，当细胞汇合度达到70%时，将培养基更换为不含胎牛血清（FBS）、添加100 nM地塞米松（DEX）与1%青霉素-链霉素溶液的DMEM/F12培养基，即同步化培养基（SCM）。使用添加100 nM地塞米松的SCM完成初始昼夜节律同步化后，将细胞分别置于GVCM与HVCM中培养24小时，记为T0时刻。在T0时刻后，分别于32h（T8，对应上午时段）与44h（T20，对应下午时段）采集细胞，对应得到Grain-am、Grain-pm、Hay-am与Hay-pm四个处理组，该两个时间点分别定义为上午与下午时段。最后，将四个处理组的细胞置于-80℃快速冷冻，用于后续针对H3K27ac、H3K9ac与H3K4me3相关的CUT&Tag测序实验，每个文库设置3个混合生物学重复样本。