ChIP-Seq of NSL3 and MBD-R2 in Drosophila melanogaster S2 cells to investigate the function of Non-Specific Lethal (NSL) complex
ChIP-Seq of NSL3 and MBD-R2 in Drosophila melanogaster S2 cells to investigate the function of 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. 30ml 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 2.5M 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%. 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. 3 ul antibody were added to the chromatin and rotated overnight at 4C. Protein A/G-Sepharose was blocked with 0.1% BSA and 1 mg/ml of short dsDNA (15 bp) for 1 hour. 20 ul of the sepharose was added to the chromatin/antibody mixture and incubated for 3 hours in 4C. The sepharose beads were then washed four times for 15 minutes in RIPA buffer, once in LiCl buffer (0.25 M LiCl, 10 mM Tris at pH 8, 1 mM EDTA, 0.5% NP-40, 0.5% DOC), and two times in TE buffer. The beads were incubated at 65C overnight in TE buffer to reverse the cross-link. The sample was then treated 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
NSL3