GSM9544222: K562_BCOR_8h5phIAA_H3K4me3_1; Homo sapiens; ChIP-Seq
Cells were cross-linked using PBS including 1% formaldehyde for 10 minutes. Cross-linked samples were quenched using 125 mM glycine, followed by a PBS wash and snap freezing SDS buffer (100 mM NaCl, 0.5% SDS, 5 mM EDTA, 50 mM Tris-HCl (pH 8.0), protease inhibitors (CLAP and 0.1 mM PMSF), and 0.2% NaN3. Next, cross-linked samples were IP buffer containing: 2x SDS buffer and 1 x Triton Dilution buffer (5% Triton X-100, 5 mM EDTA, 100 mM Tris-HCl (pH 8.6), 100 mM NaCl, 0.2% NaN3, 1 x CLAP, 0.1 mM PMSF). Sonication was performed using a Bioruptor (Diagenode) or PIXUL® Multi-Sample Sonicator (Active Motif). Then, the samples were precleared using 1 mg pre-equilibrated Dynabeads Protein G (Invitrogen) for 20 minutes and subsequently ChIP reactions were conducted in 1 ml IP buffer and incubated overnight at 4 °C with 5% mouse spike-in control chromatin and 2 µg of either of the following antibodies: anti-GFP (ab290, Abcam), IgG (I8141, Sigma), anti-KDM2B (09-846, Merck), anti-H2AK119ub (D27C4, Cell Signaling Technology), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (C15410196, Diagenode), anti-H3K27me3 (C15410069, Diagenode) and anti-H3K36me3 (C15410192, Diagenode). Next, ChIP reactions were incubated for 3h with 25 ml pre-equilibrated Dynabeads Protein G (invitrogen) at 4°C and subsequently washed with Mixed Micelle Wash buffer (150 mM NaCl, 20 mM Tris-HCl (pH 8.1), 5 mM EDTA, 5.2% sucrose, 1% Triton X-100, 0.2% SDS, 0.2% NaN3, 3 washes), buffer 500 (50 mM Hepes-KOH (pH 7.5), 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.2% NaN3; 2 washes), LiCl Wash buffer (250 mM LiCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.2% NaN3; 2 washes) and TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA; 1 wash). Finally, chromatin was eluted from the beads using Elution Buffer (0.1 M NaHCO3, 0.1% SDS) and reverse cross-linked overnight at 65 degrees. Finally, 2 ml RNase A (10mg/ml; ThermoFisher) was added and samples were incubated for 2 hours at 37 degrees followed by addition of 2 ml Proteinase K (20mg/ml; ThermoFisher) and incubation for 2 hours at 55 degrees. Finally, the DNA was extracted using a PCR purification kit (Qiagen). The purified DNA samples were analyzed by sequencing. ChIP-seq libraries were prepared with the KAPA Hyper Prep Kit (Roche Sequencing and Life Sciences) following the manufacturer's instructions. The resulting DNA fragment libraries were subjected to a sequencing on an Illumina NextSeq500 and generated the paired-end reads FASTQ files, using the manufacturer's instructions.
GEO
GSM9544222_r1