title: GSM7116206: BC2121 RPMI H3K27Ac; Homo sapiens; ChIP-Seq
library_construction_protocol: Monocytes were resuspended in RPMI culture medium containing 1% formaldehyde (Sigma Aldrich) and incubated for 10 min at RT. Formaldehyde was quenched using 125 mM glycine, which was incubated for 5 min at RT. Monocytes were washed twice in cold PBS supplemented with protease inhibitor cocktail (Roche). Cell pellets were snap frozen in liquid nitrogen and stored at -70°C until further use. Cells and nuclei were lysed using Magna ChIP™ Cell Lysis buffer and Nuclear Lysis buffer (Merck Millipore) according to manufacturer's instructions. Lysates were sonicated using a Bioruptor pico sonicator (Diagenode; 10 cycles, 30 s on, 30 s off, at 4°C) in a density of 15*106 cells/mL and spun down. Sheared chromatin was collected and fragment sizes were determined by DNA purification using MinElute Reaction Cleanup Kit (Qiagen) followed by gel electrophoresis. Immunoprecipitation was performed using the Magna ChIP™ A Chromatin Immunoprecipitation Kit (Merck Millipore) according to manufacturer's instructions. In short, sonicated lysates derived from 5*105 cells were incubated overnight with 1 μg H3K4me3 antibody or H3K27ac (Diagenode) and protein A magnetic beads at 4 °C on a rotating platform. Protein A beads-antibody/chromatin complexes were washed with buffers provided by the Magna ChIP kit for 5 min at 4 °C on a rotating platform. Chromatin was eluted by incubation in Elution buffer supplemented with proteinase K (Magna ChIP kit, Merck Millipore) for 2 h at 62 °C, shaking, followed by 10 min at 95 °C. DNA was purified and eluted in 25 uL milliQ. ChIP-seq libraries were prepared using the Kapa Hyper Prep Kit according to manufacturer's instructions, with the following modifications. For adaptor ligation of each sample, 2.5 mL of the NEXTflex adaptor stock (600 nM, Bioo Scientific) was used. Libraries were amplified with 12-15 PCR cycles followed by a double post-amplification clean-up to ensure proper adapter removal. Samples were analyzed for purity using a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent). Libraries were paired-end sequenced to a read length of 42 bp on an Illumina NextSeq500.