GSM5292594: TS116_09_INPUT; Homo sapiens; ChIP-Seq
ChIP for histone marks or SOX4 transcription factor were performed as previously described in (Lee et al., 2006) using either 1x107 or 5x107 AML patient cells respectively that were thawed and immediately cross-linked. Cells were cross-linked at room temperature using 1% formaldehyde. For SOX4 ChIP-seq a previous step of Gold cross-linking (Diagenode) was included, as per manufacturer's instructions. After 10 minutes the reaction was quenched by incubation for 10 minutes with 0.125M glycine. Cell pellets were washed twice with cold PBS and 1x107 or 5x107 cells were used per ChIP. Briefly, nuclear lysates were sonicated using a Bioruptor Plus (Diagenode) for 6-10 min at high, 30s ON, 30s OFF settings. Immunoprecipitation was performed overnight at 20rpm and 4°C, with 10μl magnetic beads (Dynabeads (Protein G), Invitrogen, Carlsbad, CA) per 1μg antibody per 1x107 cells. Antibodies used were: acetyl-H3K27 (ab4729, Abcam, Cambridge, UK; 1μg per ChIP), anti-trimethyl H3K27 (9733S, Cell Signalling; 1μg per ChIP), anti-monomethyl-H3K4 (ab8895, Abcam; 1μg per ChIP) anti-trimethyl H3K9 (ab8898, Abcam; 1μg per ChIP) or SOX4 (CS-129-100, Diagenode; 15μl per ChIP). After washing five times 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 at 65°C for 30min with elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) vortexing frequently. Cross-linking was then reversed by incubation at 65°C for 16 hours. RNAse A (1mg/ml) and proteinase K (20mg/ml) were used to eliminate any RNA or protein from the samples. Finally DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and precipitated with ethanol (adding two volumes of ice-cold 100% ethanol, glycogen (20μg/μl) and 200mM NaCl) for at least 1 hour at -80°C. Pellets were washed with 70% ethanol and eluted in 30μl 10mM Tris-HCl pH 8.0. Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode) or NEBNext UltraII (New England Biolabs) and 1ng ChIP to the manufacturer's instructions. Fragments of 200–800 bp were selected using AMPure beads (Beckman Coulter, Pasadena, CA) and quantified by quantitative PCR with a KAPA Library Quantification Kit (Kapa Biosystems, Woburn, MA). Sequencing was performed using either a Novaseq6000 or a NextSeq500 desktop sequencer system (Illumina) with 75-bp, paired-end high output generating 18–58 million reads per sample for the histone marks ChIP-seq or 96 million reads for the SOX4 ChIP-seq experiments.