Sample information curated by ChIP-Atlas

Antigen

Antigen Class
No description
Antigen
NA

Cell type

Cell type Class
Cell line
Cell type
S2
Source
Oregon R
Developmental Stage
late embryonic stage

Attributes by original data submitter

Sample

ENA first public
2012-06-19
ENA last update
2018-03-08
External Id
SAMEA1467044
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
Submitter Id
E-MTAB-1084:Cell culture 4
broker name
ArrayExpress
cell line
S2
cell type
embryonic cell
common name
fruit fly
sample name
E-MTAB-1084:Cell culture 4
sex
male

Sequenced DNA Library

library_name
DNA extract 8
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

dm6

Number of total reads
149789918
Reads aligned (%)
82.0
Duplicates removed (%)
66.9
Number of peaks
37739 (qval < 1E-05)

dm3

Number of total reads
149789918
Reads aligned (%)
82.2
Duplicates removed (%)
66.0
Number of peaks
39254 (qval < 1E-05)

Base call quality data from DBCLS SRA