GSM3591587: P5 Ctrl Rep2 K27ac; Homo sapiens; ChIP-Seq
Cells were fixated in 1% formaldehyde in PBS for 10 min at room temperature followed by quenching with glycine (final concentration of 0.125M) to stop the crosslinking reaction. Fixated cells were washed with PBS and harvested in 1 ml SDS Buffer (50mM Tris-HCl (pH 8), 100mM NaCl, 5mM EDTA (pH 8.0), 0.2% NaN3, 0.5% SDS, 0.5 mM phenylmethylsulfonyl fluoride) and centrifuged for 6 min at 250g. The pelleted nuclei were lysed in 1.5 ml ice-cold IP Buffer (67mM Tris-HCl (pH 8), 100mM NaCl, 5mM EDTA (pH 8.0), 0.2% NaN3, 0.33% SDS, 1,67% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride) and sonicated (Diagenode, Biorupter) to an average length of 200-500 bp (between 15 and 20 cycles, high intensity). For preparation of chromatin from human skeletal muscle biopsies, frozen biopsies (20-40 mg) were thawed on ice and chopped into small pieces (between 1-3 mm3). The tissue was fixated in 0.5% formaldehyde in PBS for 7.5 min at room temperature followed by quenching with glycine (0.125M). The fixated tissue was washed with PBS before resuspension in 1 ml of IP buffer and homogenization by douncing until the tissue was completely disassociated. The chromatin was sonicated (Diagenode, Biorupter) to an average length of 200-500 bp (between 20 and 30 cycles, high intensity). Before starting the ChIP experiment, chromatin was cleared by centrifugation for 30 min at 20,000g. For each ChIP, 2-10 ug DNA was combined with 2.5 ug antibody and incubated with rotation at 40C for 16 hours. The following antibodies were used for ChIP: H3K27ac (Ab4729), H3K4me1 (Ab8895), H3K4me3 (CST-9751S), H3 (Ab1791). Immunoprecipitation was performed by incubation with Protein G Sepharose beads (GE healthcare) for 4 hrs followed by three washes with low-salt buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 150 mM NaCl) and two washes with high-salt buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 500 mM NaCl). Chromatin was de-cross-linked in 120 ul 1%SDS and 0.1M NaHCO3 for 6h at 650C, and DNA was subsequently purified using Qiagen MinElute PCR purification kit For library preparation and sequencing, 3-10 ng of immunoprecipitated DNA was used to generate adaptor-ligated DNA libraries using the NEBNext® Ultra DNA library kit for Illumina (New England Biolabs, E7370L) and indexed multiplex primers for Illumina sequencing (New England Biolabs E7335). DNA libraries were sequenced on a Illumina HiSeq 2000 by 50‐bp single‐end sequencing at the National High‐Throughput Sequencing Centre (University of Copenhagen, Denmark)