Cut&Run H3K27ac inVivo HFD BA 10mg

For CUT&RUN against H3K27ac in mouse liver
samples, we utilised the EpiCypher inc CUTANA ChIC/CUT&RUN kit (EpiCypher, SKU: 14-952
1048) 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 cell
suspension was then centrifuged at 1000 xg for 5 minutes at 4°C. The samples were
subsequently washed with 1 ml PBS (Sigma D8537) supplemented with cOmplete protease
inhibitor cocktail (Roche, CO-RO SKU 11697498001) and 10 mM sodium butyrate (Sigma
Aldrich, 303410), followed by centrifugation under the same conditions. The following steps
were performed in presence of protease inhibitors and sodium butyrate. Washed cells were
resuspended in 1 ml of NIB with 0.3% IGEPAL CA-630 (Sigma, I8896) and incubated on ice
for 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 mM
Tris-HCl (Carl Roth, Art.-Nr. 9090.3) pH 7.5, 60 mM KCl (Carl Roth, Art.-Nr. 6781.3), 11 mM
CaCl2 (Carl Roth, Art.-Nr. CN93.1), 5 mM NaCl (Carl Roth, Art.-Nr. 3957.2), 5 mM MgCl2 (Carl
Roth, Art.-Nr. KK36.2), 250 mM sucrose (Sigma, S0389), 1 mM 1,4-Dithiothreitol (Carl Roth,
Page 37
Art.-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 step
was performed with 1 ml of NIB, and after centrifugation, and the nuclei resuspended in 100
μl of NIB without IGEPAL CA-630. The nuclei were counted, and 0.5 x 106 nuclei were used
for 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). Sequencing
libraries were prepared using NEBNext Ultra II library preparation procedure (New Englad
Biolabs, E7645L) with modifications according to NGS Library preparation of CUTANA
ChIC/CUT&RUN kit (EpiCypher, SKU: 14-1048, Version 1.0), and then assessed for quality
and quantity by BioAnalyzer (Agilent). Generated libraries were sequenced on an Illumina
NovaSeq instrument (150 bp, paired-end)