Illumina Genome Analyzer II sequencing; EcR recruits and stimulates dMi-2 remodeling activity to constrain transcription of hormone-regulated genes
EcR recruits and stimulates dMi-2 remodeling activity to constrain transcription of hormone-regulated genes
S2 cell lines were maintained under standard conditions.  The conditions for S2 cell incubation for all experiments were set to six hours treatment with 1μM 20HE (in 5% EtOH). S2 nuclear and whole cell extracts were prepared and immunoprecipitated as described (Kunert et al., 2009; Meier et al., 2012). For immunoprecipitation of endogenous Mi-2, Protein G Sepharose was preincubated for 1hr with 1% fish skin gelatine and buffer D (0.2 mg/mL BSA in 20 mM Hepes KOH, pH 7.6 100 mM KCl 1.5 mM MgCl2, 0.2 mM EDTA, 20% (v/v) Glycerol, 0.1 mM DTT, protease inhibitors) at room temperature. Antibodies were incubated with pre-blocked Protein G sepharose for 1 hr at 4°C. Beads were washed twice with buffer D / 0.02% NP40 and transferred to low-binding tubes. 30ul of Protein G Sepharose were incubated with nuclear extract for 3 hrs at 4°C. The beads were washed five times with buffer D/0.02% NP40 and eluted with 40ul 2x SDS loading buffer. ChIP samples were sequenced on an Illumina IIx Genome Analyzer according to the manufacturer's instructions.
Experimental Factor: Ecdysone
compound
Experimental Factor: 1
dose