title: GSM4671870: Mll3KOMll4KI_D4_H3K27ac; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 30 million fixed cells were swelled in cold buffer containing 5mM PIPES (pH 8.0), 85mM KCl, 1% NP-40 and protease inhibitors for 20 min, and centrifuged at 2,000 g for 5 min at 4°C. Nuclei were resuspended with 3 mL TE buffer (pH 8.0), and subjected to sonication. Sheared chromatin was clarified by centrifugation at 4,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with 150mM NaCl, 0.1% SDS, 1% Triton-X100 and protease inhibitors. 2% of the total material was set aside as input and 20 ng of spike-in chromatin (Active Motif, #53083) was added to each ChIP material. For each ChIP material, 2 - 8 µg of target antibodies and 1.5 μg of spike-in antibody (anti-H2Av, Active Motif, #61686) pre-bound with protein A Dynabeads (ThermoFisher) were added and incubated on a rotator at 4°C overnight. Beads were then collected on a magnetic rack and washed twice with 1 mL cold RIPA buffer, once with 1 mL cold RIPA buffer containing 500mM NaCl, twice with 1 mL cold LiCl buffer and once with 1 mL TE buffer. Finally, beads were eluted with 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 100 μg Proteinase K at 65°C overnight. Samples were then purified using QIAquick PCR purification kit (Qiagen) and eluted in 50 μL 10mM Tris-HCl. Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).
identifiers.namespace: GEO
identifiers.id: GSM4671870