|
| Status |
Public on Aug 04, 2019 |
| Title |
CBR_3 |
| Sample type |
SRA |
| |
|
| Source name |
BMDM
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: wild type
cell type: bone marrow-derived macrophages
treatment: IL4+CBR-5884
|
| Treatment protocol |
Differentiated BMDMs were seeded in DMEM high glucose (Gibco), 10% low endotoxin FBS (Gibco), 2 mM L-glutamine (Lonza), 100 U/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma), 50 μg/ml β-mercaptoethanol (Gibco) over night. The next morning, they were pre-treated with 30 µM of the PHGDH inhibitor CBR-5884 (AxonMedchem) or solvent. Thereafter, 10 ng/ml IL-4 (Peprotech) was added and cells were harvested 24h later.
|
| Growth protocol |
Bone marrow, isolated from femur, tibia and humerus was differentiated for 6 days on petri dishes. Differentiation medium consisted of DMEM high glucose (Gibco), 10% low endotoxin FBS (Gibco), 2 mM L-glutamine (Lonza), 100 U/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma), 50 μg/ml β-mercaptoethanol (Gibco) supplemented with either 15 ng/ml CSF1 (Peprotech) or 10–20 % L929 conditioned supernatant. Three days later, non-adherent cells were removed and cells were split 1:2 in fresh medium. On day 6, differentiated BMDM (96% of the cells were positive for F4/80 and CD11b) were washed, dislocated gently with a cell scraper, and seeded in the indicated medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy Plus Micro Kit was used to isolate total RNA according to manufacturer's instructions. Quality control of RNA samples was performed using RNA 6000 Nano Kit on an 2100 Bioanalyzer (Agilent).
Sequencing libraries were prepared at the Core facility Genomics, Medical University of Vienna using the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina according to manufacturer’s protocols (New England Biolabs). Libraries were QC-checked on a Bioanalyzer 2100 (Agilent) using a High Sensitivity DNA Kit for correct insert size and quantitated using Qubit dsDNA HS Assay (Invitrogen).
Pooled libraries had an average length of 330-360bp and were sequenced on a NextSeq500 instrument (Illumina) in 1 x 75 bp sequencing mode.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
The raw reads in fastq format have been aligned to the mouse mm10 genome with Bowtie2/Tophat2 on the combined reference transcriptome from Ensembl 75 and UCSC mm10.
The identified transcripts are further assembled and analyzed for differential analysis with the pipeline Cufflinks.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *_transcripts.gtf: GTF files include FPKMs.
|
| |
|
| Submission date |
Aug 03, 2018 |
| Last update date |
Aug 05, 2019 |
| Contact name |
Thomas Weichhart |
| E-mail(s) |
thomas.weichhart@meduniwien.ac.at
|
| Organization name |
Medical University of Vienna
|
| Department |
Center for Pathobiochemistry and Genetics
|
| Street address |
Waehringerstraße 10
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE118119 |
Inverse data-driven modelling and multiomics analysis reveals PHGDH as a metabolic signature of M2 macrophages |
|
| Relations |
| BioSample |
SAMN09765554 |
| SRA |
SRX4507277 |
