GSM4826520: TS98_8_54_T2; Homo sapiens; ChIP-Seq
ChIP was performed using anti-H3K27Ac (ab4729, Abcam, Cambridge, UK). 10^8 cells (THP-1) or 107 cells (primary AML samples) were used for each precipitation using the method described by Lee et al. (2006). Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature before the reaction was quenched with 0.125M glycine. Cell pellets were washed twice with PBS and nuclear lysates sonicated for 6 cycles using a Bioruptor® Pico (Diagenode, Liege, Belgium). 10μg of antibody bound to 100μl of magnetic beads (Dynabeads Protein G, Invitrogen, Carlsbad, CA) was added to each sample and immunoprecipitation performed overnight on a rotator at 4°C and 20rpm. After five washes with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl), chromatin IP-bound fractions were extracted by incubating for 15 min at 65°C with elution buffer (50mM TrisHCl pH8, 10mM EDTA, 1% SDS). Crosslinking was then reversed by incubation at 65°C for 6 hours. RNaseA (1mg/ml) and proteinase K (20mg/ml) were added to eliminate RNA and protein from the samples. DNA was extracted using phenol:chloroform:isoamyl alcohol and precipitated by adding 2 volumes of ice-cold 100% ethanol, glycogen (20μg/μl), 200mM NaCl and freezing at -80°C for at least 1hr. Pellets were washed with 70% ethanol and eluted in 50μl 10mM TrisHCl pH8.0. Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode). 200-800bp fragments were selected using AMPure beads (Beckman Coulter, Brea, CA) and quantified by qPCR with a KAPA Library Quantification Kit (Kapa Biosystems, Basel, Switzerland). Sequencing was performed using a NextSeq desktop sequencing system (Illumina) with 75bp, paired-end high output generating 65-80M (THP-1_S and THP-1_R) and 34-45M reads per sample (primary AML samples).