|
| Status |
Public on May 28, 2019 |
| Title |
IRF9KO STAT1 90 min IFNg rep2 |
| Sample type |
SRA |
| |
|
| Source name |
IRF9KO STAT1 90 min IFNg, BMDM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8-10 weeks
treatment: IFNg
chip antibody: Stat1 (E23) Santa Cruz sc-346, RRID:AB_632435
genotype: Irf9-/-
cell type: bone marrow derived macrophages
|
| Treatment protocol |
1.5 x 10^7 cells were seeded with 90 % confluency on 15 cm dishes, respectively. The next day cells were stimulated for 90 minutes either with with 10 ng/ml murine interferon gamma (IFN-γ; eBioscience) or 250 IU/mL of IFNβ (PBL interferon source).
|
| Growth protocol |
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow isolated from femurs and tibias of 8- to 10-week-old mice. Femur and tibia were separated by cutting at the knee joint. Bones were flushed with Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) using a 5-mL syringe and a 25-gauge needle. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% of fetal calf serum (FCS) (Sigma-Aldrich), recombinant M-CSF, 100 units/ml penicillin, and 100 ng/ml streptomycin (Sigma-Aldrich). Cells were kept at 37°C and 5% CO2 and differentiated for 10 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed 2x with ice cold PBS and crosslinked for 10 minutes at room temperature in 1% formaldehyde PBS (thermos fischer #28906). The cross-linked chromatin was sheared by sonication, providing fragments of 200 - 1400 base pairs in length.Protein-DNA complexes were selectively immunoprecipitated using STAT1,STAT2 and IRF9 antibodies. The immunoprecipitated complexes were washed to remove non-specifically bound chromatin, the protein-DNA cross-link was reversed and proteins were removed by proteinase K digest. Four IPs were pooled for each sample of the two biological replicates, in order to obtain enough DNA precipitated. The DNA associated with the complex was furthermore purified and used for ChIP-seq.
For library generation, the NEBNext Ultra II DNA Library Prep Kit for Illumina from NEB was used according to the manufacturer’s protocol. The chromatin quality was analyzed on an Agilent 2100 Bioanalyzer and a size selection of 200-1400bp was carried out.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Raw reads were processed using the AQUAS TF pipeline (https://github.com/kundajelab/chipseq_pipeline;based off Encode phase-3;-idr_thresh 0.01): including alignment against the Mus musculus mm10 genome using BWA (v0.7.13), deduplication using Picard MarkDuplicates (v1.126), Peak Calling using macs2 (v2.1.1) and spp (v1.13).
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak formatted peak sets for each replicate as called by macs2; narrowPeak formatted optimal peak sets for each condition using SPP peak calling for both replicates and combined using idr analysis (idr_threshold 0.01)
|
| |
|
| Submission date |
Jun 06, 2018 |
| Last update date |
May 28, 2019 |
| Contact name |
Ekaterini Platanitis |
| Organization name |
University of Vienna
|
| Department |
Department of Microbiology, Immunobiology and Genetics
|
| Street address |
Dr.-Bohr-Gasse 9
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE115433 |
STAT1, STAT2 and IRF9 transcription factor binding analysis in wild type and Irf9-/- bone marrow derived macrophages in response to type I and type II interferons |
| GSE115435 |
Irf9 function in immunity in mouse |
|
| Relations |
| BioSample |
SAMN09377575 |
| SRA |
SRX4178781 |
