CID island ChIP
CID island ChIP
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).
Experimental Factor: Immunoprecipitate
input_DNA