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
2015-06-17
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA2794491
INSDC center alias
Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna
INSDC center name
Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna
INSDC first public
2015-06-17T17:01:16Z
INSDC last update
2018-03-08T21:18:06Z
INSDC status
public
Submitter Id
E-MTAB-2972:Deckers_lab_Sep2014_STAT1_IFNb
broker name
ArrayExpress
cell type
bone marrow macrophage
common name
house mouse
genotype
wild type genotype
sample name
E-MTAB-2972:Deckers_lab_Sep2014_STAT1_IFNb
sex
female

Sequenced DNA Library

library_name
Deckers_lab_Sep2014_STAT1_IFNb
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. After 10 days the cells were used for experiment. 2.5x107 cells were seeded on a 15cm dish and treated as follows. Cells used for ChIPseq experiments with antibodies against STAT1 were treated with 250U/ml IFNb for 2 hours. Cells subjected to ChIP with antibodies against NFkB p65 or RNA polymerase II were treated with IFNb (250U/ml) and hkL (equivalent to MOI 50) for 4 hours. After treatment formaldehyde was added to the cells to a final concentration of 1% and dishes were incubated for 10 minutes at 37C. 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 (0.25%Triton X-100; 0.01M EDTA; 0.5mM EGTA; 0.01M Hepes) for 10 minutes at 4C, followed by another centrifugation step. The pellet was resuspended in wash II (0.2M NaCl; 1mM EDTA; 0.5mM EGTA; 0.01M Hepes) and kept for 10 minutes at 4C. Cells were lysed over night with lysis buffer (1% SDS; 0.05M Tris pH 8; 0.01M EDTA) at 4C. The cells were then sonicated 27 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. 5 ug antibody was added to the chromatin and rotated overnight at 4C. Protein G Dynabeads (Invitrogen) were blocked over night in 0.1% BSA and on the next day added to the chromatin/antibody mixture and incuabted for 3 hours at 4C. Beads were then washed five times for 10 minutes in RIPA buffer (0.15M NaCl; 0.05M Tris pH 8; 0.1% SDS; 0.5% NaDoc; 1% NP40), once in high salt buffer (0.5M NaCl; 0.05M Tris pH 8; 0.1% SDS; 1% NP40), once in LiCl buffer (0.25M LiCl; 0.05M Tris pH 8; 0.5% NaDoc; 1% NP40), and two times in TE buffer. Beads were then shaken in elution buffer at room temperature for 40 minutes and 20ul 4M NaCl was added to the eluted sample. Samples were incubated at 65C overnight to reverse the cross-links. Samples were then treated with proteinase K (2ul ptroteinase K, 8ul EDTA 0.5M pH 7.5 and16ul IM TRIS pH 6.5) for 60 min at 55C. 500ul Phenol/Chloroform/Isoamyl alcohol (25/24/1) was added to all samples and they were then centrifuged for 5 minutes at 16000g. The upper 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 QPCR NGS library quantification kit (KAPA).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
77258454
Reads aligned (%)
96.9
Duplicates removed (%)
30.7
Number of peaks
3230 (qval < 1E-05)

mm9

Number of total reads
77258454
Reads aligned (%)
96.8
Duplicates removed (%)
30.9
Number of peaks
3230 (qval < 1E-05)

Base call quality data from DBCLS SRA