 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 18, 2022 |
| Title |
p38i_An_IFNg rep1 (ATAC-seq_IFNg) |
| Sample type |
SRA |
| |
|
| Source name |
WT p38i_An_IFNg, BMDM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: wildtype
age: 8-12 weeks
treatment: p38i_Anisomycin_IFNg
cell type: Bone marrow derived macrophage (BMDM)
|
| Treatment protocol |
3x106 BMDM were seeded in 6cm non-treated tissue culture plates.The next day cells were stimulated for 140 minutes with Anisomycin (Sigma-Aldrich) alone, or either with 10ng/mL of murine IFNγ (a kind gift from G. Adolf, Boehringer Ingelheim, Vienna) alone for 120 minutes or with Anisomycin (20 minutes pre-treatment) followed by 120 minutes stimulation with IFNγ or with p38i inhibitor (PH-797804; Selleckchem) (60 minutes pre-treatment) and Anisomycin (20 minutes pre-treatment) followed by 120 minutes stimulation with IFNγ.
|
| 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 twice in 1 mL ice-cold PBS, by centrifugation at 1200 g for 5 min. Cells were resuspended in 1 mL cold nuclei isolation buffer (0.32 M sucrose; 3 mM CaCl2; 2mM Mg Acetate; 0.1 mM EDTA; 10 mM Tris.HCl pH 8.0; 0.6% NP-40; 1mM DTT fresh) and placed on ice for 5 minutes. Cells were centrifuged at 700g for 5 min at 4°C. Supernatant was removed, 500 µL cold nuclei isolation buffer was added and samples were kept on ice for 3 min to further increase the number of isolated nuclei. Cells were centrifuged at 700g for 5 min at 4°C. Supernatants were removed and pelleted nuclei were gently resuspended in 200 µL cold Nuclei Resuspension Buffer (NRB) (50 mM Tris.HCl pH 8.3; 40% Glycerol; 5 mM MgCl2; 0.1mM EDTA). 50.000 nuclei (5µl) were resuspended in 7.5µl H2O, 12.5 µl TD buffer and 5 µl Tn5 transposase (Illumina). Nuclei were incubated for 30 min at 37 °C, while mixing on a plate shaker at 600 rpm. DNA was purified with Qiagen MinElute columns according to the manufacturer's instructions and eluted in 13µl of nuclease-free H2O.
12.5 µl purified DNA from the previous step were mixed with 5 µl 2.5 µM I7 index primer, 5 µl 2.5 uM I5 index primer (dual indexing), 2.5 µl Evagreen and 25 µl, 2x Q5 PCR Master Mix, (NEB). An end point PCR was run as follows: 5 min 72 ºC, 1 min 98 ºC, 10sec 98 ºC (5-7 cycles) 30sec 65 ºC (5-7 cycles), 60sec 72 ºC (NEB). The reaction was purified by adding 15 µl of SPRI beads prepared by the NGS facility (MBSpure beads) and incubated for 5 min at RT. The supernatant was transferred to a fresh well. 50µl of MBSpure beads were added and mixed well, incubated for 5 min at RT, magnetized, and the supernatant was removed. The beads were washed twice by adding 150 µl of 80% EtOH. Beads were dried for 30sec and DNA was eluted in 20 µl of nuclease-free water. The quality of the libraries was checked on a bioanalyzer to further determine the size distribution. Libraries were sequenced on a NovaSeq SP PE50.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
cell nuclei
|
| Data processing |
Data were processed using the ATAC-seq pipeline from the nf-core framework (nfcore/atacseq v1.2.1; Ewels et al., 2020; https://zenodo.org/record/3965985#.YA8a8GRKjZk) in conjunction with Docker (version 20.10.8; Merkel, 2014).
The command used was " nextflow run nf-core/atacseq --input design.csv --genome GRCm38 -profile docker -r 1.2.1 --min_reps_consensus 2".
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/atacseq).
Assembly: mm10
Supplementary files format and content: Normalised genome-wide bigWig coverage files generated from merged BAM files included in the mergedReplicate folder of 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) |
| GSE199126 |
Chromatin accessibility analysis of wild type bone marrow-derived macrophages in response to Anisomycin, type II interferon and the combination of them or with p38 MAPK inhibitor PH-797804 (ATAC-Seq IFNg) |
| GSE199166 |
Stress signaling boosts interferon-induced gene transcription in macrophages |
|
| Relations |
| BioSample |
SAMN26867262 |
| SRA |
SRX14564080 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
