|
| Status |
Public on Dec 18, 2022 |
| Title |
IFNg_CREB1_rep3 (ChIP-seq) |
| Sample type |
SRA |
| |
|
| Source name |
IFNg_CREB1, BMDM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8-12 weeks
treatment: IFNg
chip antibody: CREB1
cell type: Bone marrow derived macrophage (BMDM)
|
| 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 140 minutes with Anisomycin, or with with 10 ng/ml murine IFN-γ (a kind gift from G. Adolf, Boehringer Ingelheim, Vienna) for 120 minutes alone or in combination with 1 µM p38 inhibitor (PH-797804; Selleckchem) and 20µM JNK inhibitor (SP600125; Sigma Aldrich) for 60min pre-treatment followed by 20 minutes Anisomycin pre-treatment or the combination of the two inhibitors alone.
|
| Growth protocol |
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow isolated from femurs and tibias of 8- to 12-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 (ThermoFisher #28906). The cross-linked chromatin was sheared by sonication, providing fragments of 200 - 1000 base pairs in length. Protein-DNA complexes were selectively immunoprecipitated using CREB,c-Jun and igG 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. At least three IPs were pooled for each sample of the three biological replicates, in order to obtain enough DNA precipitated. The DNA associated with the complex was furthermore purified and used for ChIP-seq.
The library generation was carried out by the VBCF NGS facility (https://www.viennabiocenter.org/vbcf/next-generation-sequencing/). The chromatin quality was analyzed on an Agilent 2100 Bioanalyzer and a size selection of 200-1000bp was carried out.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw data were processed using the ChIP-seq pipeline from the nf-core framework (nfcore/ChIP-seq v.1.2.2; Ewels et al., 2020; https://zenodo.org/record/3529400#.YA8YGmRKjZk) in conjunction with Docker (version 20.10.8; Merkel, 2014).
The command used was " nextflow run nf-core/chipseq --input design.csv --genome GRCm38 -profile docker --min_reps_consensus 2 -r 1.2.2 --narrow_peak" .
For a condensed overview of the pipeline, the bioinformatics tools used in each step and an extensive list of citations please see the pipeline homepage (https://github.com/nf-core/chipseq).
Assembly: mm10
Supplementary files format and content: Normalised genome-wide bigWig coverage files generated from merged BAM files with bamCompare from deeptools version 3.4.3 (https://zenodo.org/record/3965985#.Yh-DPejMJPZ) with --scaleFactorsMethod readCount as method to scale the samples.
Supplementary files format and content: Peak .narrowPeak file were generated from the nfcore pipeline
|
| |
|
| Submission date |
Mar 22, 2022 |
| Last update date |
Dec 18, 2022 |
| Contact name |
Laura Boccuni |
| E-mail(s) |
laura.boccuni@univie.ac.at
|
| 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 |
GPL24247 |
| Series (2) |
| GSE199127 |
c-Jun and CREB1 transcription factor binding analysis in wild type bone marrow-derived macrophages in response to type II interferon and stress |
| GSE199166 |
Stress signaling boosts interferon-induced gene transcription in macrophages |
|
| Relations |
| BioSample |
SAMN26867068 |
| SRA |
SRX14563994 |
