title: GSM2967389: PRIMARY_CHOR_A269A1_H3K27AC (CF_269); Homo sapiens; ChIP-Seq
library_construction_protocol: For H3K27ac ChIP of patient-derived tissue: approximately 100 mg of flash-frozen tissue was minced into 1-2 mm pieces and incubated in 1% formaldehyde for 15 minutes, followed by quenching with glycine (125 mM final concentration). Fixed tissue pieces were homogenized with a Tissue Tearor rotor stator homogenizer (Biospec) set to 30,000 RPM for 60 seconds. Homogenate was washed with ice-cold PBS containing 1X HALT protease inhibitor. Homogenized pellets were then resuspended in cytosolic lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL, 0.25% Triton X-100, 1X HALT protease inhibitor) and the protocol was continued as described below. For H3K27ac ChIP of chordoma cell lines: Chordoma cell lines were grown in 175 cm collagen I-coated plates (3 plates per cell line) and cross-linked by adding 1/10 of the cell culture volume of 11% formaldehyde solution (1M HEPES-KOH pH 7.5, 0.5 M EDTA pH 8.0, 0.5 M EGTA pH 8.0, 5 M NaCl, 37% formaldehyde) for 10 minutes, followed by quenching (125 mM glycine). Cells were washed in PBS and harvested by cell scraper in PBS. Cells were centrifuged at 1,350 X g for 5 min at 4°C, washed with PBS, and centrifuged again at 1,350 X g for 5 min at 4°C. Cell pellets were flash frozen and stored at -80°C until further processing. For both cell line and tissue ChIP-seq: samples were subsequently rotated end-over-end for 10 minutes at 4º C in cytosolic lysis buffer and then collected by spinning at 1,350 x g for 5 minutes. Samples were resuspended in nuclear lysis buffer (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5mM EGTA, 1X HALT protease inhibitor) and rotated end-over-end for 10 minutes at 4º C and spun at 1,350 x g for 5 minutes. Samples were resuspended in 1 mL sonication buffer ( 0.1% Na-Deoxycholate, 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1X HALT protease inhibitor) with SDS added to a final concentration of 0.5%. Chromatin was sheared by sonication using a Bioruptor water bath sonicator (Diagenode) for 25 cycles. Samples were clarified by centrifuging 10 minutes at 20,000 x g at 4º C. A 50 uL aliquot of supernatant was set aside as non-IP input control. The remaining sheared chromatin was diluted 1:5 in sonication buffer and each sample was incubated overnight at 4º C with 100 uL of Protein G Dynabeads (Life Technologies) bound with 10 µg of anti-H3K27ac antibody (abcam, #ab4729). Beads were washed while rotating end-over-end at 4º C with the following buffers: twice in sonication buffer, once in sonication buffer with NaCl added to a final concentration of 500 mM, once in LiCl wash buffer (20 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% IGEPAL, 0.5% Na-Deoxycholate), and once in TE-NaCl buffer (50 mM Tris-HCl pH 8, 50 mM NaCl). Chromatin was eluted by resuspending beads in 200 uL elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) and heating for 15 minutes at 65º C. Beads were pelleted by centrifuging at 20,000 G for 30 seconds and supernatant was collected. Elution buffer (150 uL) was also added to input chromatin samples. Samples were incubated at 65º C overnight to reverse crosslinks. Samples were then incubated with RNAse A (Thermo Scientific) for 2 hours at 37º C, followed by incubation with Proteinase K (Ambion) for 30 minutes at 55º C. DNA isolation was performed via phenol-chloroform extraction and concentrated by ethanol precipitation. Sequencing libraries were generated with the Thruplex DNAseq Single Index Kit (Rubicon) and sequenced on an Illumina NextSeq 500 with single-end, 75 bp reads. ChIP-seq was performed once for each sample.
attributes.tag: GEO Accession
attributes.value: GSM2967389