GSM8119672: liver, H3K4me3, Sham rep4; Mus musculus; ChIP-Seq
ChIP was performed on 200-400 mg of snapfrozen liver tissue. First, DynabeadsTM protein G (Thermo Fisher Scientific) were washed three times with BSA/PBS and coupled to a primary antibody against H3K27ac or H3K4me3 overnight at 4°C. Afterwards, the beads with coupled antibody were washed with BSA/PBS twice. Livers were homogenized and fixed in Farnham Lysis buffer with 1% formaldehyde, supplemented with protease inhibitor cocktail (Roche), for 10 min at RT. the material was sonicated (Bioruptor, Diagenode) at 4°C for 40 cycles (30s ON, 30s OFF) with high settings to yield 200-800 bp DNA fragments. Cell debris was removed by centrifugation at full speed for 20-30 min at 4°C. Sonication efficiency was checked (after reverse crosslinking with proteinase K for 1h at 65°C and DNA purification) on a 1.5% agarose gel. For immunoprecipitation (IP), antibody-coupled magnetic beads were added to the sonicated samples and incubated overnight at 4°C . The day after, bead-antibody-DNA complexes were washed 5 times with LiCl wash buffer (100 mM Tris-HCl; pH 7.5, 500 mM LiCl, 1% IGEPAL, 1% sodium deoxycholate) at 4°C and 2 times with TE buffer (10 mM Tris-HCl; pH 7.5, 0.1 mM EDTA) at 4°C. Chromatin was eluted by incubating the IP samples with elution buffer (1% SDS, 0.1M NaHCO3) 1h at 65°C. Samples  were incubated with proteinase K for 4h at 65°C for reverse crosslinking and DNA was purified in 50 µL elution buffer according to the manufacturer's protocol by using the Monarch PCR&DNA Cleanup kit (Biolabs). 15 ng of purified chromatin was used to generate a ChIP-Seq library according to the manufacturer's protocol using NEBNext Ultra II DNA Library Prep kit (E7645L, NEB)