GSM3597655: BJfibroblastsctrl_p63ChIP_rep2; Homo sapiens; ChIP-Seq
Crosslinked cells were quenched with glycine (125mM final) for 5 min, followed by 2 x washes in cold PBS. Nuclei were then isolated from 20 million cells as previously described (Shah et at. 2013), and chromatin  was sheared to 250bp average size using a Covaris S220. Immunoprecipitations were performed using 500µg sheared chromatin lysate and 5 µg antibodies preconjugated to Protein G beads (Invitrogen): H3K27ac (Active Motif, 39133), Flag (Sigma, M2, F1804) and hKLF4 (R&D Systems, AF3640). ChIP reactions were incubated for 16 hours at 4˚C with rotation and then washed 4x in wash buffer (50mM HEPES-HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-laurylsarcosine), followed by 1 wash in ChIP final wash buffer (1xTE, 50mM NaCl). Immunoprecipitated DNA was eluted from washed beads, reverse crosslinked overnight, purified and used to construct libraries. Sequencing libraries for ChIP experiments were prepared using NEBNext Ultra reagents (New England Biolabs). All ChIP samples and input were double-end sequenced on an Illumina NextSeq 500.
GEO Accession
GSM3597655