title: E-MTAB-652:d5
organism: Drosophila melanogaster
attributes.tag: Alias
attributes.value: E-MTAB-652:d5
attributes.tag: Broker name
attributes.value: ArrayExpress
attributes.tag: CellLine
attributes.value: Schneider S2
attributes.tag: Description
attributes.value: Protocols: S2 cells were grown in Schneider's Drosophila medium (Serva) supplemented with 10% fetal calf serum and antibiotics (0.3 mg/ml penicillin, 0.3 mg/ml streptomycin and 0.75 μg/ml amphotericin B). Cells were transfected with FuGENE® 6 Transfection Reagent (Roche) and stable lines were selected with 100 mg/ml Hygromycin-B as previously described 25 or in case of co-transfection with pCoPuro with 100mg/ml puromycin. Cells containing stably integrated arrays of Lac Operators on chromosome 3R were obtained by co-transfection with pAFS51 56 and pCoPuro, followed by subsequent cloning of the cell line. Pulse-overexpression of CID and pulse-co-overexpression of CID and HP1 were induced using the metallothionein promoter (pMT/V5 vectors) with 500 mM CuSO4 for 48 h (start day -2). At day 0 Fluorescent Activated Cell Sorting (FACS) was used to select the cells with the highest levels of GFP expression (>75% of max.) on a MoFlo sorter using 70 mm 11 diameter nozzle at 60 psi. Cells were immediately plated in fresh medium lacking inducing reagents for the chase. For HDAC inhibition cells were incubated with 75nM Trichostatin A (Cell Signaling Technology, Inc) from the beginning of CIDGFP pulse (day -2). 4x107 S2 cells in 40 ml Schneider's medium were fixed for 10 minutes at room temperature by addition of 1% formaldehyde. After 2 ice-cold washes in PBS, cells were lysed in 2.6ml of L2 buffer (L2: 1% SDS, 5mM EDTA, 50mM Tris pH8). The lysate was sonicated to fragment genomic DNA to an average length of around 500 bp, and then diluted 10-fold in DB (DB: 0.5 % NP40, 5 mM EDTA, 200 mM NaCl, 50 mM Tris pH8). Anti-mouse IgG M280 magnetic beads were coated with mouse monoclonal anti-GFP antibody (clone 496) by rotating together for 6 hours at 4ºc (80mg antibody for 160 ml M280 slurry), followed by washing in DB. For each immunoprecipitation, 160 ml slurry of anti-GFP-coated beads were added to the sonicated lysate from 107 cells and rotated overnight at 4ºC. Beads were washed once quickly and then 3x for 5 minutes on ice in WB (WB: 0.1% SDS, 1% NP40, 2mM EDTA, 500mM NaCl, 20mM Tris pH8), followed by 3x for 5 minutes on ice in TE (TE: 1mM EDTA, 10mM Tris pH8). Precipitated chromatin was eluted from the beads, and fixation reversed, by incubation overnight at 65ºc in 60ml EB (EB: 2% SDS, 1mM EDTA, 10mM Tris pH8). DNA was purified using a Qiagen MinElute clean-up kit, and eluted in 30ml (elution buffer: 10mM Tris pH8.5).
attributes.tag: INSDC center name
attributes.value: MAX-PLANCK-INSTITUTE
attributes.tag: INSDC first public
attributes.value: 2011-07-15T17:00:23Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T15:26:30Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: SRA accession
attributes.value: ERS032714
attributes.tag: Sample Name
attributes.value: ERS032714
attributes.tag: Sex
attributes.value: male
attributes.tag: Title
attributes.value: d5