ChIP-seq  profiling of RNA Polymerase II in Drosophila melanogaster S2 cells depleted of the non-specific lethal (NSL)  complex
ChIP-seq  profiling of RNA Polymerase II in Drosophila melanogaster S2 cells depleted of the non-specific lethal (NSL)  complex
S2 cells were grown in Schneiders Media (Invitrogen) supplemented with 10% of heat-inactivated bovine fetal serum to a density of 1 million cells per ml. Cells at a density of 1 million per ml were transfected with 10 ug dsRNA against NSL1, NSL3 or GFP using Lipofectamine RNAiMAX (Invitrogen) according to manufacturers instructions. The cells were harvested after 6 days. 30 ml cell suspension were spun down and the cell pellet was resuspended in 1 ml of cross-linking solution (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). 37% formaldehyde was added to a final concentration of 1.8%. The tube was rotated at room temperature for 15 minutes and the reaction was quenched with 2.5 M glycine at a final concentration of 125 mM for 5 minutes. The fixed cells were washed 3 times for 5 minutes each with rinse buffer 1 (10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and 5 times for 5 minutes each with rinse buffer 2 (10 mM HEPES pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Finally, the cells were washed 3 times in RIPA buffer (25 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.1% DOC). The cells were sonicated 30 x 20 seconds using a Branson 250 sonicator at pulse 40, intensity 3%, followed by Covaris sonication. The chromatin should be sheared into fragments with average length of 200 bp. Samples were centrifuged at maximum speed and the supernatant was used for the subsequent steps. To reverse cross-linking, samples were incubated at 65C overnight in TE buffer, followed by a treatment with RNaseA (0.2 mg/ml) for 30 min at 37C, and with Proteinase K (0.05 mg/ml) for 2 hours at 50C. Finally, DNA was purified using Minelute columns (Qiagen).
Experimental Factor: IMMUNOPRECIPITATE
Input
Experimental Factor: RNAi
GFP