title: GSM1254212: P286_H3K27AC; Homo sapiens; ChIP-Seq
library_construction_protocol: Primary tissue samples from DLBCL tumors, normal lymph node, or normal tonsil that were flash-frozen and stored in OCT were collected in 10 slices at 20 ¼m. Frozen biopsy specimens of newly diagnosed, previously untreated primary DLBCLs with >80% tumor involvement and known transcriptional subtyping (Monti et al., 2012) were obtained according to Institutional Review Board (IRB)-approved protocols  (Brigham & Women Hospital, and Dana-Farber Cancer Institute). A waiver to obtain informed consent was granted by the local IRBs because otherwise discarded tissue was used. After two washes and spin downs in PBS (300g, 5 min), tissue slices were then cross-linked with 1.1% formaldehyde in PBS for 10 min. The formaldehyde was quenched by adding 60 ul/ml of 2.5 M glycine. Glycine and fixative were removed with two washes in cold PBS. Samples were homogenized with a Dounce homogenizer using 10 strokes with pestle A and 10 strokes pestle B in a solution of 10 mM Tris HCl pH8, 10 mM NaCl, 3 mM MgCl2, and 1% NP40. Sample was then transferred to a 15 ml conical tube and rotated in the cold room for 60 min. After this incubation, nuclei were spun down at 1350g for 10 min and transferred to a 3.5 ml tube in 50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.7% SDS. Samples were then sonicated on ice for 10 cycles at 30 s (18 W) with 60 s on ice between cycles. All lysis and sonication buffers contain Complete protease inhibitor cocktail (Roche). Sonicated lysates were cleared by centrifuging at 20,000g for 10 min and incubated overnight on a spinning wheel at 4oC with magnetic beads prebound with antibody after diluting sample to 0.1% SDS final. 100 _l of Dynal magnetic beads per sample (Invitrogen) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were loaded with 10 _g of each antibody overnight at 4oC. Antibodies used were as follows: Histone H3K27Ac: Abcam ab4729 ; BRD4: Bethyl A301-985A. Beads were washed three times with sonication buffer, one time with sonication buffer supplemented with 500 mM NaCl, one time with LiCl wash buffer (20 mM Tris pH 8.0, 1 mM EDTA, 250 mMLiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and once with TE. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8, 10mM EDTA, and 1% SDS). Cross-links were reversed overnight at 65oC. RNA and protein were digested with 0.2mg/mL RNase A for two hr followed by 0.2 mg/ml Proteinase K for one hr. DNA was purified with phenol chloroform extraction and ethanol precipitation.
attributes.tag: GEO Accession
attributes.value: GSM1254212