cell_culture_1
Drosophila melanogaster
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. 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 2.5 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%. 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). The libraries were prepared following Illumina's TruSeq ChIP sample prep kit (http://www.illumina.com/products/truseq_chip_sample_prep_kit.ilmn)
ENA-FIRST-PUBLIC
2013-10-30T17:01:19Z
ENA-LAST-UPDATE
2018-03-08T16:29:47Z
External Id
SAMEA2078457
INSDC center name
European Bioinformatics Institute (EBI)
INSDC first public
2013-10-30T17:01:19Z
INSDC last update
2018-03-08T16:29:47Z
INSDC status
public
Submitter Id
E-MTAB-1705:cell_culture_1
broker name
ArrayExpress
cell line
S2
cell type
embryonic cell
common name
fruit fly
genotype
wild type
sample name
E-MTAB-1705:cell_culture_1
scientific_name
Drosophila melanogaster
sex
male