GSM5823214: 1033_H3K27ac; Homo sapiens; ChIP-Seq
OCT-compount frozen blocks from CIT cohort () were frozen-sectioned and stained with hematoxylin and eosin. Pathology review was performed to confirm the tumour stage and to select tumour areas, in order to enhance neoplastic content (estimated at 30 to 95%, median tumour cell content = 65%). For tumours with sufficient material, tumour-enriched areas were macrodissected from the frozen block and manually finely ground in a mortar. Frozen ground tumour tissue was further homogenized using a tube pestle or the TissueLyser II system (Qiagen). Disrupted tissue was then processed using the reagents from the iDeal ChIP-seq Kit for Histones (Diagenode), according to the manufacturer's instructions. Tumour samples with optimal chromatin fragment size (200-500 bp) were immunoprecipitated using the iDeal ChIP-seq Kit for Histones (Diagenode).Magnetic immunoprecipitation of sheared DNA-chromatin complexes (500ng) was performed overnight using a rabbit polyclonal histone H3K27acetyl ChIP Grade antibody (ab4729, Abcam), H3K27me3 (Active Motif, ref. 39155), and H3K9me3 (Active Motif, ref. 39161). DNA purification was performed using iPure magnetic beads. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7cycles ONLY). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.