Sample information curated by ChIP-Atlas

Antigen

Antigen Class
No description
Antigen
NA

Cell type

Cell type Class
Blood
Cell type
Macrophages
MeSH Description
The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)

Attributes by original data submitter

Sample

ENA first public
2016-01-25
ENA last update
2018-03-09
ENA-CHECKLIST
ERC000011
External Id
SAMEA3432778
INSDC center alias
Institute of Animal Breeding & Genetics, University of Veterinary Medicine Vienna
INSDC center name
Institute of Animal Breeding & Genetics, University of Veterinary Medicine Vienna
INSDC first public
2016-01-25T17:02:28Z
INSDC last update
2018-03-09T01:23:49Z
INSDC status
public
Submitter Id
E-MTAB-3597:Deckers_lab_May2015_STAT1_Y701F_untreated
broker name
ArrayExpress
cell type
bone marrow macrophage
common name
house mouse
genotype
Stat1 Y701F mutant
sample name
E-MTAB-3597:Deckers_lab_May2015_STAT1_Y701F_untreated

Sequenced DNA Library

library_name
Deckers_lab_May2015_STAT1_Y701F_untreated
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Bone marrow derived macrophages (BMDM) were obtained by culture of mouse bone marrow in DMEM +10% FCS +10% L-cell-derived conditioned medium containing colony-stimulating factor 1 and antibiotics. The cells were used for the experiments after 10 days in culture. 15x106 cells were seeded on a 15 cm dish and treated with 250 U/mL of mouse recombinant IFNβ for 2 h or left untreated before harvesting. The ChIP was performed using antibody against mouse STAT1 (clone E-23, Santa Cruz). After treatment with IFNβ, formaldehyde was added to the cells to a final concentration of 1% and dishes were incubated for 15 minutes at 37°C. The reaction was quenched by adding 2.5 M glycine to a final concentration of 125 mM for 10 minutes. The fixed cells were washed with PBS and scraped. After centrifugation for 10 minutes at 600g cells were washed with wash I buffer (0.25%Triton X-100; 0.01M EDTA; 0.5 mM EGTA; 0.01 M Hepes) for 10 minutes at 4°C, followed by another centrifugation step. The pellet was resuspended in wash II buffer (0.2M NaCl; 1mM EDTA; 0.5 mM EGTA; 0.01 M Hepes) and kept for 10 minutes at 4°C. Cells were lysed over night with lysis buffer (1% SDS; 0.05M Tris pH 8; 0.01M EDTA) at 4°C. Cells were then sonicated 25 x 30 seconds using a Bioruptor sonicator. This resulted in chromatin fragments with an average length of 300 bp. Samples were centrifuged twice at 16.000g for 15 min and the supernatant was used for the subsequent steps. 1 µL of antibody was added to 25-30 µg of chromatin and rotated overnight at 4°C. Protein G Dynabeads (Invitrogen) were blocked over night in 0.1% BSA and on the next day added to the chromatin/antibody mixture and incubated for 3 hours at 4°C. Beads were then washed for 10 minutes in following buffers consecutively: RIPA buffer (0.15 M NaCl; 0.05 M Tris pH 8; 0.1% SDS; 0.5% NaDoc; 1% NP40), high salt buffer (0.5 M NaCl; 0.05 M Tris pH 8; 0.1% SDS; 1% NP40), LiCl buffer (0.25 M LiCl; 0.05 M Tris pH 8; 0.5% NaDoc; 1% NP40), and two times in TE buffer (1 M Tris pH 8, 0.5 M EDTA). Beads were then shaken in elution buffer (1 mL 10% SDS, 0.5 mL 1M NaHCO3, 50 µL 1M DTT in 5 mL of H2O) at room temperature for 40 minutes and 20 µl 4M NaCl was added to the eluted sample. Samples were incubated at 65°C overnight to reverse the cross-links. Samples were then treated with proteinase K (2 µl ptroteinase K, 8 µl EDTA 0.5 M pH 7.5 and 16 µl 1M TRIS pH 6.5) for 60 min at 55°C. 500 µl Phenol/Chloroform/Isoamyl alcohol (25/24/1, Sigma) was added to all samples and they were then centrifuged for 5 minutes at 16000g. The aqueous phase was used for DNA precipitation. 5-10 ng of DNA precipitate was used for the generation of sequencing libraries using the KAPA library preparation kit for Illumina systems. Libraries were quantified with a Bioanalyzer dsDNA 1000 assay kit (Agilent) and a Q-PCR NGS library quantification kit (KAPA).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
55270086
Reads aligned (%)
96.4
Duplicates removed (%)
21.1
Number of peaks
286 (qval < 1E-05)

mm9

Number of total reads
55270086
Reads aligned (%)
96.3
Duplicates removed (%)
21.1
Number of peaks
242 (qval < 1E-05)

Base call quality data from DBCLS SRA