GSM6533619: H3K-2416N_Control_Refed (ChIP); Mus musculus; ChIP-Seq
Chromatin was prepared from frozen tissue using truChIP Chromatin Shearing Tissue Kit (Covaris). Chromatin was sheared using Diagenode Bioruptor Pico for 30 s ON/ 30 s OFF 10 cycles in SDS lysis buffer (1% SDS, 10 mM EDTA pH 8.1; 50 mM Tris-HCl, pH 8.1) supplemented with protease inhibitor cocktail. Input were decrosslinked overnight at 65C in elution buffer (0.1M NaHCO3; 0.1% SDS; 200mM NaCl) followed by treatment with RNAseA (0.125ug/ml) and Proteinase K (0.1mg/ml). Chromatin was purified using QIAQuick PCR Purification Kit and quantified using Qubit (dsDNA HS). Dynabeads Protein A (Invitrogen) were preincubated with 4 ug of H3K27Ac (ab4729, Abcam) or Rabbit anti-GATA4 Antibody (A303-503A, Fortis Life Sciences) in ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1mM EDTA, pH 8.1; 16.7 mM Tris-HCl, pH 8.1; 167 mM NaCl + proteinase inhibitor) for 1.5 hours at room temperature. 7 -– 10 ug of chromatin were incubated overnight with the corresponding antibody-bound beads with rotation for immunoprecipitation (IP). IP samples were washed with low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA pH 8.1; 20mM Tris-HCl pH 8.1; 150 mM NaCl), followed by high salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA pH 8.1; 20mM Tris-HCl pH 8.1; 500 mM NaCl), LiCl wash buffer (250 mM LiCl; 1% deoxycholic acid; 1mM EDTA pH 8.1, 10 mM Tris-HCl pH 8.1; 1% Igepal) and finally 2 washes with TE Buffer (1 mM EDTA pH 8.1; 10 mM Tris-HCl pH 8.1) (Ferrari et al. 2017). IP samples were de-crosslinked and purified as described for the input above. Libraries were made with KAPA DNA HyperPrep kit