GSM5155741: 15209X09: H3K27ac ChIP-seq, vehicle control rep #1; Mus musculus; ChIP-Seq
ChIP assays were performed from approximately 107 cells per experiment, according to previously described protocol with slight modifications (Rada-Igelsias, Nature 2010).  Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was quenched by addition of glycine to a final concentration of 0.125 M. Chromatin was sonicated to an average size of 100-600 bp, using Bioruptor Pico (Diagenode). 50–75 µl of protein G dynal beads (Invitrogen) were used to capture 3–5 µg of antibody in phosphate citrate buffer pH 5.0 (2.4 mM citric acid, 5.16 mM Na2HPO4) for 30 min at 27 °C. Antibody bead complexes were rinsed two times with PBS and added to sonicated chromatin and rotated at 4 °C overnight. Ten per cent of chromatin was reserved as 'input' DNA. Magnetic beads were washed 5 times with 500mM LiCl wash buffer and once with 50mM NaCl+TE and chromatin eluted, followed by reversal of the crosslinkings and DNA purification. Resultant ChIP DNA was dissolved in TE.fter reversal of cross-links, libraries were prepared with an NEBnext DNA Library Prep kit (NEB, E7370L). Adaptor-ligated DNA was then size-selected and purified with AMPure XP beads (Beckman Coulter) and PCR amplified as above (see RNA-seq library prep). HA epitope tag (for p53-HA) abcam ab91110; H3K9me3 (active motif 39161); H3K27ac (abcam 4729). After reversal of cross-links, libraries were prepared with an NEBnext DNA Library Prep kit (NEB, E7370L). Adaptor-ligated DNA was then size-selected and purified with AMPure XP beads (Beckman Coulter) and PCR amplified as above (see RNA-seq library prep).