|
| Status |
Public on Dec 18, 2022 |
| Title |
An rep1 (RNA-seq) |
| Sample type |
SRA |
| |
|
| Source name |
WT An, BMDM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: wildtype
age: 8-12 weeks
treatment: Anisomycin
cell type: Bone marrow derived macrophage (BMDM)
|
| Treatment protocol |
1 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 (Sigma-Aldrich) alone, or either with 10 ng/ml murine interferon gamma (IFN-γ; A kind gift from G. Adolf, Boehringer Ingelheim, Vienna) or 250 IU/mL of IFNβ (PBL interferon source) alone for 120 minutes or with Anisomycin (20 minutes pre-treatment) followed by 120 minutes stimulation with either IFN types.
|
| 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 |
total RNA |
| Extraction protocol |
Cells were harvested after treatments and RNA was isolated using the Qiagen AllPrep DNA/RNA Mini Kit according to the manufacturer's guidelines. RNA-seq libraries were prepared with TruSeq Stranded mRNA LT sample preparation kit (Illumina) using Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library amounts were quantified using Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using Experion Automated Electrophoresis System (Bio-Rad). For sequencing, libraries were pooled, diluted and sequenced on an Illumina HiSeq 3000 instrument using 50 bp single-read chemistry.
Library was performed at the Biomedical Sequencing Facility (https://www.biomedical-sequencing.org/) with PolyA capture to obtain mRNA molecules
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Adapter trimming via trimmomatic (using catalogue of known contaminant sequences)
Alignment via Bowtie 1 to cDNA (from Ensembl)
Transcript quantification via bitSeq https://github.com/databio/rnapipe/blob/master/src/rnaBitSeq.py
For visualization only: adapter trimming via trimmomatic (using catalogue of known contaminant sequences)
Alignment via TopHat 2 to DNA
Generation of BigWig / TrackHub with rnaTopHat (https://github.com/databio/rnapipe/blob/master/src/rnaTopHat.py)
Differential expression was assessed with DESeq2 v. 1.24.0 (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8).
Assembly: GRCm38 (mm10)
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: comma-separated-value (csv) files holding the name of every gene and their basemean, Log2FC, log fold change standard error (lfcSE), p-value and adjusted p-value for comparison with UT samples of three biological replicates.
|
| |
|
| 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 |
GPL21493 |
| Series (2) |
| GSE199128 |
Transcription profile analysis of wild type bone marrow derived-macrophages in response to Anisomycin, type I and type II interferons and the combination of them |
| GSE199166 |
Stress signaling boosts interferon-induced gene transcription in macrophages |
|
| Relations |
| BioSample |
SAMN26867118 |
| SRA |
SRX14563886 |
