Cut&Run H3K9ac inVivo HFD Control

For CUT&RUN against H3K27ac in mouse liversamples, we utilised the EpiCypher inc CUTANA ChIC/CUT&RUN kit (EpiCypher, SKU: 14-9521048) according to the manufacturer’s instructions with minor modifications.For in vivo experiments, approximately 200 mg of liver tissue was homogenized in DMEM(Gibco, 41966-029) using a potter tube and a glass pestle with 12 strokes. The resulting cellsuspension was then centrifuged at 1000 xg for 5 minutes at 4°C. The samples weresubsequently washed with 1 ml PBS (Sigma D8537) supplemented with cOmplete proteaseinhibitor cocktail (Roche, CO-RO SKU 11697498001) and 10 mM sodium butyrate (SigmaAldrich, 303410), followed by centrifugation under the same conditions. The following stepswere performed in presence of protease inhibitors and sodium butyrate. Washed cells wereresuspended in 1 ml of NIB with 0.3% IGEPAL CA-630 (Sigma, I8896) and incubated on icefor 5 minutes. The nuclear suspension was then centrifuged at 1000 xg for 5 minutes at 4°C.The resulting nuclear pellet was washed with 1 ml of nucleus isolation buffer (NIB) (15 mMTris-HCl (Carl Roth, Art.-Nr. 9090.3) pH 7.5, 60 mM KCl (Carl Roth, Art.-Nr. 6781.3), 11 mMCaCl2 (Carl Roth, Art.-Nr. CN93.1), 5 mM NaCl (Carl Roth, Art.-Nr. 3957.2), 5 mM MgCl2 (CarlRoth, Art.-Nr. KK36.2), 250 mM sucrose (Sigma, S0389), 1 mM 1,4-Dithiothreitol (Carl Roth,Page 37Art.-Nr. 6908.3), 10 mM sodium butyrate (Sigma Aldrich, 303410)) without IGEPAL CA-630,and the nuclei were subsequently filtered through a 30 μm strainer. An additional washing stepwas performed with 1 ml of NIB, and after centrifugation, and the nuclei resuspended in 100μl of NIB without IGEPAL CA-630. The nuclei were counted, and 0.5 x 106 nuclei were usedfor the subsequent procedure, either in step 7 for ACLY ChIC (Version 1.0 EpiCypher, SKU:14-1048) or in step 14 for H3K9ac and H3K27ac ChIC (Version 3.3 EpiCypher, SKU: 14-1048)of the manufacturer's protocol.For each reaction, 2 μg of antibody was used (Diagenode C15410196). Sequencinglibraries were prepared using NEBNext Ultra II library preparation procedure (New EngladBiolabs, E7645L) with modifications according to NGS Library preparation of CUTANAChIC/CUT&RUN kit (EpiCypher, SKU: 14-1048, Version 1.0), and then assessed for qualityand quantity by BioAnalyzer (Agilent). Generated libraries were sequenced on an IlluminaNovaSeq instrument (150 bp, paired-end)针对小鼠肝脏样本中H3K27ac的CUT&RUN实验，我们采用了EpiCypher公司的CUTANA ChIC/CUT&RUN试剂盒（EpiCypher，货号：14-9521048），严格遵循制造商说明书进行操作，仅做少量微调。体内实验环节：取约200 mg肝脏组织，使用Potter匀浆管与玻璃研杵，在DMEM培养基（Gibco，货号：41966-029）中以12次往复的方式进行匀浆。所得细胞悬液于4℃、1000×g条件下离心5分钟。随后使用添加了cOmplete蛋白酶抑制剂混合物（Roche，CO-RO货号：11697498001）与10 mM丁酸钠（Sigma-Aldrich，货号：303410）的1 ml PBS（Sigma D8537）洗涤样本，再以相同离心条件进行分离。后续所有操作均在蛋白酶抑制剂与丁酸钠存在的体系中完成。洗涤后的细胞重悬于1 ml含0.3% IGEPAL CA-630的NIB缓冲液中，置于冰上孵育5分钟。随后将细胞核悬液以4℃、1000×g离心5分钟，收集细胞核沉淀。使用1 ml不含IGEPAL CA-630的细胞核分离缓冲液（NIB）洗涤沉淀，该缓冲液配方为：15 mM Tris-HCl（Carl Roth，货号：9090.3，pH 7.5）、60 mM KCl（Carl Roth，货号：6781.3）、11 mM CaCl₂（Carl Roth，货号：CN93.1）、5 mM NaCl（Carl Roth，货号：3957.2）、5 mM MgCl₂（Carl Roth，货号：KK36.2）、250 mM蔗糖（Sigma，货号：S0389）、1 mM 1,4-二硫苏糖醇（Carl Roth，货号：6908.3），第37页、10 mM丁酸钠（Sigma-Aldrich，货号：303410）。之后将细胞核悬液通过30 μm细胞滤网过滤。再使用1 ml NIB缓冲液进行一次额外洗涤，离心后将细胞核重悬于100 μl不含IGEPAL CA-630的NIB缓冲液中。对细胞核进行计数后，取0.5×10⁶个细胞核用于后续实验流程：可选择执行ACLY ChIC实验的第7步（EpiCypher 1.0版，货号：14-1048），或执行H3K9ac与H3K27ac ChIC实验的第14步（EpiCypher 3.3版，货号：14-1048），均遵循制造商提供的实验方案。每个反应体系使用2 μg抗体（Diagenode，货号：C15410196）。测序文库构建采用NEBNext Ultra II文库制备流程（New England Biolabs，货号：E7645L），并参照CUTANA ChIC/CUT&RUN试剂盒（EpiCypher，货号：14-1048，1.0版）的NGS文库制备方法进行优化调整。随后使用生物分析仪（BioAnalyzer，Agilent）对文库的质量与浓度进行检测评估。最终构建的文库在Illumina NovaSeq测序仪上进行双端测序，读长为150 bp。