title: GSM4460519: Pooled Input; Mus musculus; ChIP-Seq
library_construction_protocol: Frozen lung tissue was pulverized, followed by cross-linking of proteins to DNA with 37% formaldehyde, and then incubation with 10X glycine to stop the reaction. Cross-linked tissue was homogenized, and centrifuged at 1,500 rpm for 5 min at 4°C. The cell pellet was resuspended with cell lysis buffer (PBS with 0.5 mM EDTA and 0.05% Triton X-100), incubated on ice for 15 min, followed by centrifiugation at 5,000 rpm for 5 min at 4°C. The cell pellet was then resuspended with nuclear lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl at pH 8.1) and incubated on ice for 10 min. Chromatin was sonicated with a bioruptor (Diagenode, Denville, NJ) to obtain fragment sizes of 100-300 bp for ChIP-seq. ChIP was performed by incubating sheared chromatin with Magna ChIPTM Protein A+G magnetic beads, and antibodies against H3K27ac overnight at 4°.The next day, the beads were washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 150 mM NaCl) high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 500 mM NaCl), lithium chloride wash buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl pH 8), and TE buffer (20 mM Tris-HCl pH 8 and 1 mM EDTA) for 5 minutes each at 4°. Immunoprecipitated DNA was recovered from the beads by incubation with ChIP elution buffer (1% SDS, 1 mM EDTA, 50 mM NaHCO3, and 50 mM Tris-HCl pH 8) and proteinase K for 2 hours at 62° with shaking, followed by incubation at 95° for 10 minutes. DNA was purified using the QIAquick PCR Purification kit (Qiagen) and DNA concetration was measured using a NanoDrop spectrophotometer (Thermo Scientific). Sequencing libraries were prepared using the NexFlex Bioo Kit Option 2 protocol using 1.7-10 ng of DNA per sample.