P1034_CM_C2P22_UDID031_ChIPH3K27ac_R02L01S02
Detailed methods are found in doi 2024.04.10.588874. 5-15 x 10^6 formaldehyde crosslinked cells were lysed and sonicated in 110ul of SDS Lysis Buffer (0.5% SDS, 50mM Tris-HCl pH 8.0, 20mM EDTA, 1x cOmplete Protease Inhibitor Cocktail (Sigma)) using Covaris E220 Focused-ultrasonicators (Covaris) for 14 cycles, 1 min per cycle, duty cycle 5. For each sample, H3K27ac antibody (Abcam ab4729, lot GR00324078) was coupled for 4 hours to 40ul of 1-to-1 mix Protein G and Protein A Dynabeads (Thermo Scientific) and used for overnight chromatin immunoprecipitation in IP buffer (1% Triton X-100, 0.1% DOC, 1x TE buffer, 1x cOmplete Protease Inhibitor Cocktail). Beads with immunoprecipitated chromatin were washed with 150ul  of following buffer, four times with RIPA Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.0, 300mM NaCl, 0.1% DOC), two times in RIPA High Salt Buffer (0.1% SDS, 1% Triton X-100, 1mM EDTA, 20mM Tris-HCl pH 8.0, 500mM NaCl, 0.1% DOC), twice in LiCl Buffer (250mM LiCl, 0.5% NP-40, 0.5% DOC, 1mM EDTA, 10mM Tris-HCl pH 8.0) and twice in 1X TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Next samples were eluted in 150 ul of Direct Elution Buffer (0.1% SDS, 10mM Tris-HCl pH 8.0, 5mM EDTA) and reverse crosslinked by incubation for 15 min at 65C with rotation and subsequent incubation with 5 ul RNAse (Sigma) for 1h at 37C and Proteinase K Solution (20 mg per mL, Thermo Fisher Scientific) for 1h at 55C. After reverse crosslinking samples were purified with 2X Agencourt AMPure XP DNA beads (Beckman Coulter), eluted in 30 ul of H2O and Qubit (Thermo Scientific) quantified. Libraries were generated using KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Real Time Library Amplification Kit (KAPA Biosystems) following manufacturers manual. Libraries were barcoded using TruSeq RNA Indexes (Illumina), size selected for 300 bp to 500 bp, and sequenced with either 100 bp paired-end or 150 bp paired-end reads on an Illumina HiSeq 4000.