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)