GSM9769961: Mll4-AID_MyoD-ER-WT_ETOH_T7; Mus musculus; ChIP-Seq
For ChIP-Seq, cells were cross-linked with 1.5% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 10 million fixed cells were resuspended in ice cold buffer containing 5mM PIPES, pH 7.5, 85mM KCl, 1% NP-40 and protease inhibitors, incubated on ice for 15 min, and centrifuged at 500 g for 5 min at 4°C. Nuclei were resuspended with 1mL buffer containing 50mM Tris-HCl, pH8.0, 10mM EDTA, 0.1% SDS and protease inhibitors, and subjected to sonication. Sheared chromatin was clarified by centrifugation at 13,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with 1% Triton-X100, 0.1% sodium deoxycholate and protease inhibitors. 2% of the mixture was set aside as input and 20ng of spike-in chromatin (Active Motif, #53083) was added to the rest. For each ChIP, 4 - 10 µg of target antibodies and 2μg of spike-in antibody (anti-H2Av, Active Motif, #61686) were added and incubated on a rotator at 4°C overnight. ChIP samples were added with 50μL prewashed protein A Dynabeads (ThermoFisher) and incubated for 3h at 4°C. Beads were then collected on a magnetic rack and washed twice with 1mL cold RIPA buffer, twice with 1mL cold RIPA buffer containing 300mM NaCl, twice with 1mL cold LiCl buffer and once with PBS. Beads were then eluted with 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 20μg Proteinase K at 65°C overnight. Input sample volumes were adjusted to 100μL with the elution buffer. Samples were then purified using QIAquick PCR purification kit (Qiagen) and eluted in 30 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). For nascent RNA-Seq, cells were incubated with 0.5 mM 5-ethynyl uridine (EU) for 30 minutes prior to harvest. Total RNA was isolated, and 1 µg was used for ribosomal RNA depletion using the NEBNext rRNA Depletion Kit (NEB, E7405L). Newly synthesized RNA was then selectively labeled and enriched via biotin conjugation using the Click-iT Nascent RNA Capture Kit (Thermo Fisher, C10365) following the manufacturer's guidelines. The enriched RNA fraction was reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher, 11754050). cDNA libraries were subsequently prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770) according to standard protocols.
GEO
GSM9769961_r1