Cut&Run H3K27ac inVivo Chow BA 30mg

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操作，仅做少量修改。体内实验中，取约200 mg肝脏组织，使用波特氏匀浆管与玻璃研杵，以12次往复研磨在达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium，DMEM，Gibco，货号41966-029）中进行匀浆。所得细胞悬液于4℃、1000×g条件下离心5分钟。随后使用1 mL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）的磷酸盐缓冲液（Phosphate Buffered Saline，PBS，Sigma D8537）洗涤样本，再以相同条件离心。后续所有步骤均在蛋白酶抑制剂与丁酸钠存在的条件下进行。将洗涤后的细胞重悬于1 mL含0.3% IGEPAL CA-630（Sigma，货号I8896）的细胞核分离缓冲液（Nucleus Isolation Buffer，NIB）中，冰浴孵育5分钟。随后将细胞核悬液以4℃、1000×g离心5分钟，所得细胞核沉淀用1 mL不含IGEPAL CA-630的细胞核分离缓冲液（NIB：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（1,4-Dithiothreitol，Carl Roth，货号6908.3）（第37页）；10 mM丁酸钠（Sigma Aldrich，货号303410））洗涤，之后将细胞核通过30 μm细胞筛过滤。再用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，取0.5×10^6个细胞核用于后续流程：既可用于ACLY ChIC实验的第7步（EpiCypher 1.0版，货号14-1048），也可用于H3K9ac与H3K27ac ChIC实验的第14步（EpiCypher 3.3版，货号14-1048），均遵循制造商的实验方案。每个反应体系使用2 μg抗体（Diagenode，货号C15410196）。测序文库制备采用NEBNext Ultra II文库制备流程（New England Biolabs，货号E7645L），并参考EpiCypher CUTANA ChIC/CUT&RUN试剂盒（货号14-1048，1.0版）的NGS文库制备方案进行修改。随后通过BioAnalyzer（Agilent）对文库的质量与浓度进行评估。所得文库在Illumina NovaSeq测序仪上进行150 bp双端测序。