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Sample GSM2348279

Query DataSets for GSM2348279

Status

Public on Oct 18, 2016

Title

BMDM_Irrad3_4h

Sample type

SRA

Source name

BMDM_Irrad_4h

Organism

Mus musculus

Characteristics

cell type: BMDM
strain: C57BL/6
infection: Dead (Irradiated) Mtb
time: 4 hours
replicate: 3

Treatment protocol

Log-phase cultures were collected, washed twice with PBS+0.05% Tween80 (PBS-T), and resuspended in Dulbecco's PBS (DPBS) before measuring the absorbance at 600 nm and plating CFUs. 1 ml aliquots in 2 ml tubes were irradiated at room temperature at 2.5x104 Gy using a Cs137 irradiator. Inactivation of the bacteria was verified by inoculating 100 ml in liquid and solid media and incubating for 6 weeks. Dead bacteria were 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

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.

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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

BG3_0
BMDM_all_RawCounts.txt

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

Submission date

Oct 17, 2016

Last update date

May 15, 2019

Contact name

Nuria Andreu

E-mail(s)

[email protected]

Organization name

LSHTM

Street address

Keppel Street