ERX088872	SAMEA1467043	ChIP-seq__profiling_of_RNA_Polymerase_II_in_Drosophila_melanogaster_S2_cells_depleted_of_the_non-specific_lethal_(NSL)__complex	ChIP-Seq	GENOMIC	ChIP	0	Application_Read	Forward	1	Illumina_HiSeq_2000	ERA119082	ERS127450	ERP001369	PRJEB2979	2020-06-25T00:33:17Z	Organism taxonomy_id="7227" taxonomy_name="Drosophila melanogaster"	Alias=E-MTAB-1084:Cell culture 3	Broker name=ArrayExpress	CellLine=S2	CellType=embryonic cell	Description=Protocols: 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. 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).	INSDC center alias=MPI for Molecular Genetics, Berlin, Germany	INSDC center name=MPI for Molecular Genetics, Berlin, Germany	INSDC first public=2012-06-19T10:09:21Z	INSDC last update=2018-03-08T15:44:37Z	INSDC status=public	SRA accession=ERS127450	Sample Name=ERS127450	Sex=male	Title=Cell culture 3