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| Status |
Public on Oct 18, 2016 |
| Title |
BMDM_Live3_4h |
| Sample type |
SRA |
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| Source name |
BMDM_Live_4h
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| Organism |
Mus musculus |
| Characteristics |
cell type: BMDM
strain: C57BL/6
infection: Live Mtb
time: 4 hours
replicate: 3
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| Treatment protocol |
Mid-log phase M. tuberculosis H37Rv were washed twice with PBS-T, once with DPBS, diluted in DMEM+10 ng/ml M-CSF and added to BMDM cells at a concentration of ∼2.5 × 106 CFU/flask (MOI of 5). After 4 h of infection at 37°C in 5% CO2, macrophages were treated with 200 mg/L amikacin for 1 h and washed twice with DPBS to eliminate any extracellular bacteria. Lastly, 6 ml of complete DMEM+10 ng/ml M-CSF was added to each flask. Samples were immediately processed for RNA extraction
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| Growth protocol |
Bone marrow cells were flushed from the femur and tibia of 8 to 10 week old female C57BL/6 mice and differentiated into macrophages for 7 days in DMEM (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Biosera) and 20 ng/ml M-CSF (PeproTech). On day 4, cells were fed with an additional 10 ml of media. After 7 days in culture, cells were washed with DPBS and 5×106 cells were seeded into 25 cm2 tissue culture flasks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Macrophages were lysed by adding 4 M GTC solution (4 M guanidine thiocyanate, 0.5 % sodium N–Lauroylsarcosine, 0.1M β–mercaptoethanol, 0.5 % Tween 80), and the lysate homogenized using a QIAshredder column (Qiagen). RNA was extracted by BCP phase partitioning and isopropanol precipitation, cleaned with 75% ethanol and treated with Turbo DNAse (Ambion, Life Technologies). DNA contamination was checked by PCR, and the RNA was further cleaned using an RNeasy MinElute Cleanup kit (Qiagen).
Approximately 500 ng of RNA from each sample was used to prepare individually barcoded strand-specific RNA-seq libraries. rRNA and mitochondrial RNA was removed using Ribo-Zero Gold Removal Magnetic Kit for human/mouse/rat (Epicentre, now Illumina) following the manufacturer's instructions. mRNA was purified using Agencourt RNAClean XP kit. Libraries were constructed using ScriptSeq™ Complete Kit
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
BEF3_0
BMDM_all_RawCounts.txt
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| Data processing |
reads were aligned to the Ensembl mouse genome version mm10 (GRCm38) using STAR 2.4.2a (Parameters: --outFilterMultimapNmax 20 --lnIntronMax 1000000 –alnSJoverhangMin 8 –alnSJDBoverhangMin 1)
Uniquely aligned reads in BAM format were annotated against the protein-coding mRNA regions using SeqMonk v.0.33.1 platform (Babraham Bioinformatics, Cambridge UK)
Raw counts per mRNA, strand-specific, merging isoforms were quantified using SeqMonk
Differential expression was analyzed in SeqMonk using the R (version 3.2.2) package DESeq2
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: tab-delimited text files include raw counts for each sample
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| Submission date |
Oct 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Nuria Andreu |
| E-mail(s) |
nuria.andreum@gmail.com
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| Organization name |
LSHTM
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| Street address |
Keppel Street
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| City |
London |
| ZIP/Postal code |
WC1E 7HT |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE88801 |
Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis |
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| Relations |
| BioSample |
SAMN05913739 |
| SRA |
SRX2248676 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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