GSM5066842: 17787T H3K27ac ChIP-seq; Homo sapiens; ChIP-Seq
Xenograft tumors and patient surgical specimens were digested with 1X collagenase/hyaluronidase (Stem Cell Technologies), minced, and passed through a 100μM filter. Cells were double crosslinked with 0.25M disuccinimidyl glutarate followed by 1% formaldehyde. ChIP-seq was performed as previously described3. Briefly, 2X crosslinked chromatin was sonicated on a Diagenode Bioruptor for 15 cycles 30s ON/30s OFF on the low power setting. Cells were precleared using 100μL of Magna-ChIP Protein A+G magnetic bead slurry (Millipore) before overnight incubation with 10μg ChIP antibody conjugated to100μL beads. Beads were washed once with IP Wash Buffer I, twice with High Salt Wash Buffer, once with IP Wash Buffer II, and twice with TE buffer. For buffer recipes refer to Fontanals-Cirera, Hasson et al3. Beads were eluted with 1% SDS at 65 degrees Celsius with agitation and reverse crosslinked overnight with high salt with RNase treatment (Roche). The next day, DNA underwent Proteinase K treatment at 42 degrees Celsius before elution using QIAquick PCR purification kit (QIAGEN).  DNA was run on an Agilent High Sensitivity DNA chip using an Agilent Technologies 2100 Bioanalyzer for quality control. H3K27ac ChIP-seq antibodies were purchased from abcam (#ab177178). Total RNA was extracted using the QIAGEN Rneasy Mini Kit. For ChIP-seq, antibody bound DNA was purified using the QIAGEN QIAquick PCR cleanup kit. For ChIP-seq, DNA underwent end repair, poly-adenylation, and barcoding using enzymes from New England Biolabs. ChIP libraries were amplified 12-15 PCR cycles using the 2X KAPA HiFi DNA HotStart ReadyMix before quality assessment by running on a High Sensitivity chip using a Bioanalyzer 2100 (Agilent) followed by a Qubit fluorometer (Thermo Fisher). The Mount Sinai Oncological Sciences Sequencing Core Facility prepared poly-A-capture RNA-seq libraries using the TruSeq RNA Library Prep Kit v2 (Illumina) according to manufacturer's instructions.