title: iPSC-RPE H3K27Ac: Sample c1f8a568-0130-4c20-b0c3-5f87f894b8b8
design: For H3K27ac, 5-15 x 106 formaldehyde crosslinked iPSC-RPE cells were lysed and sonicated in 110 µl 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 of H3K27ac antibody (Abcam ab4729, lot GR00324078) was coupled for 4 hours to 20µl of Protein G 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 six times with 150 µl  of ChIP wash buffer (50mM HEPES pH 8.0, 1% NP-40, 0.7% DOC, 400mM LiCl, 1mM EDTA, 1x cOmplete™ Protease Inhibitor Cocktail) and twice and 1X TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Next samples were eluted in 150 µl of ChIP Elution Buffer (1% SDS, 10mM Tris-HCl pH 8.0, 1mM EDTA) and reverse crosslinked by incubation for 15 min at 65˚C with rotation and subsequent incubation with 5 µl RNAse (Sigma) for 1h at 37˚C and Proteinase K Solution (20 mg/mL, Thermo Fisher Scientific) for 1h at  55˚C. After reverse crosslinking samples were purified with 2X Agencourt AMPure XP DNA beads (Beckman Coulter), eluted in 30 µl 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). Libraries were sequenced on an Illumina HiSeq 4000 to an average of 40 M 150 bp Paired-End reads per sample.
attributes.tag: alignment_software
attributes.value: BWA-MEM