characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-06-17T17:01:16Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:22:11Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2794495
characteristics.characteristic: External Id
characteristics.text: Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna
characteristics.characteristic: INSDC center name
characteristics.text: 2015-06-17T17:01:16Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:22:11Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS556473
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2972:Deckers_lab_Sep2014_Pol2_IFNb
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: bone marrow macrophage
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002476
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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).
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: Deckers_lab_Sep2014_Pol2_IFNb
characteristics.characteristic: title