Illumina HiSeq 2500 sequencing; Genome-wide profiling of H3K27ac (ChIP-seq) in IMR90 cells
Genome-wide profiling of H3K27ac (ChIP-seq) in IMR90 cells
Cells were cultured to confluency and treated for 90 min with 1μM dexamethasone or vehicle control (ethanol). Chromatin was fixed at room temperature for 3 min with 1% formaldehyde. Approximately three million fixed IMR90 cells were lysed in 300 μl chromatin lysis buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 1% SDS, 3% Triton X-100, 5 mM EDTA, 0.2% NaN3) supplemented with 3x protease inhibitors (Roche complete protease inhibitor cocktail) on ice for 10 minutes. Lysates were then diluted 3x with dilution buffer (50 mM Tris-HCl pH 8.6, 100 mM NaCl, 5 mM EDTA, 0.2% NaN3) and homogenized ten times with a syringe (271/2 gauge). The lysate was then aliquoted (200 μl) into 1.5 ml TPX polymethylpentene tubes (Diagenode) and sheared at 4 °C in a Bioruptor Pico for 2x10 cycles.  Chromatin Immunoprecipitation: The ChIP was performed using the Diagenode Auto Histone ChIPseq kit on an IPstar SX-8G compact automated system (Diagenode) using their indirect method (Ag + Ab → Beads). 1 μg of Diagenode ChIPseq grade rabbit polyclonal antibody (H3K27Ac; pAb-196-050) were used per ChIP. After de-crosslinking, RNAseA and proteinase K digestion, the DNA was isolated using ChIP DNA concentrator columns (Zymo Research D5205) according to the manufacturer's instructions.
Experimental Factor: vehicle control
stimulus
Experimental Factor: H3K27ac
immunoprecipitate