title: GSM9033808: Lung_wt_e15.5_Input_rep2; Mus musculus; ChIP-Seq
library_name: GSM9033808
library_construction_protocol: Lungs from E15.5 mouse embryos were cross-linked in 1% formaldehyde prepared in phosphate-buffered saline (PBS) for 10 min at room temperature and stopped by adding glycine to a final concentration of 125 mM. Samples were then washed once with PBS, snap-frozen in liquid nitrogen and kept at −80 °C until use. Lungs were disrupted with a Dounce homogenizer in Nucleus Isolation Buffer (20 mM HEPES, 250 mM sucrose, 3 mM MgCl2, 0.25% Nonidet P-40, 3 mM ß-mercaptoethanol and protease inhibitors), equilibrated on ice for 20 min, and filtered on cell strainer (70 μm) to eliminate debris. Nuclei were lysed in 50 mM Tris–HCl pH8.0, 10 mM EDTA, 1% SDS and protease inhibitors for 10 min on ice. Chromatin was then fragmented by sonication in a Covaris M220 Focused Ultrasonicator to obtain an average DNA size of 200–500 bp. Fragmented chromatin (20 μg) was incubated overnight at 4°C with 5 μl of antibody: FLAG (Sigma-Aldrich, F1804). Immunoprecipitation products were isolated with Dynabeads protein G (ThermoFisher), washed in successive saline buffers, and reverse cross-linked in 200 mM NaCl overnight at 65 °C. Samples were then digested with Proteinase K (0.12 mg/ml) and RNaseA (0.04 mg/ml) in 40 mM Tris–HCl pH8.0, 10 mM EDTA for 2 h at 50 °C. The NEBNext Ultra II DNA library prep kit from Illumina (New England Biolabs) was used to prepare DNA libraries, according to manufacturer's instructions, from 2 ng of fragmented ChIP DNA, followed by the addition of NEBNext adaptors and purification with AxyPrep Mag PCR Clean-up kit (Axygen).
identifiers.namespace: GEO
identifiers.id: GSM9033808_r1