Illumina Genome Analyzer II sequencing; Glucocorticoid receptor profiling in U2OS cells expressing GRalpha or GRgamma isoform
Glucocorticoid receptor profiling in U2OS cells expressing GRalpha or GRgamma isoform
Approximately 10 million cells were treated with 0.01% ethanol vehicle or 1 uM dexamethasone for 1.5 h. Chromatin was sheared with a Bio-ruptor water bath sonicator (Diagenode) to produce fragments of 100-200 bp. Protein-G coupled magnetic beads (Active- Motif) were preincubated for 1 h with GR-antibody (N499) before chromatin was added and incubated for an additional 2-4 h while rotating at 4C. Subsequently beads were washed three times with 10 mM TrisaHCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 5% (vol/vol) glycerol, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 0.5 mg/uL BSA, followed by three additional washes with 20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate.
Experimental Factor: immunoprecipitate
GR gamma isoform