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| Status |
Public on Dec 07, 2019 |
| Title |
D_CTL24 |
| Sample type |
SRA |
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| Source name |
JD_Positive_monocyte-derived macrophages_Control_24h
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| Organism |
Bos taurus |
| Characteristics |
cow id: D
johne's disease status: Positive
cell type: peripheral blood mononuclear cells (PBMCs)
cell subtype: monocyte-derived macrophages
ex vivo infection or control: Control
time point: 24h
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| Treatment protocol |
MAP ex vivo infections were performed for incubation periods of 1h, 4h, 8h, and 24h at a multiplicity of infection of 10 bacteria for each macrophages. Two uninfected control flasks were also collected at 4 h and 24 h. To stop the infection, cells were washed twice with PBS and harvested by adding 1.8 mL of RLT buffer(Qiagen) to each culture flask. Lysed cells were conserved at −80°C until required for RNA extraction.
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| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from 700 mL of blood drawn from the jugular vein using two blood bags containing citrate–phosphate–dextrose–adenine anticoagulant (Animal Blood Resources International, Dixon, CA, USA). PBMCs were isolated using a Ficoll gradient and monocytes were is by adherence. PBMCs were seeded in CELLSTAR TC T-75 flasks (Greiner Bio-One North America Inc, Monroe, NC, United States) at a density of 4 × 107 cells per flask with RMPI 1640 medium (Wisent, Saint-Bruno, Quebec, Canada) supplemented by 10% bovine heat-inactivated heterologous serum from JD-negative cows and a mix of L-glutamine (Wisent) and GlutaMax (Life Technologies, Burlington, Ontario, Canada) and antibiotics/antimycotics (Wisent). After an incubation period of 2 h in a humid atmosphere at 39°C with 5% CO2, non-adherent cells were removed and cells were gently washed with complete media to only keep adherent cell (monocytes). After overnight incubation, remnants of non-adherent cells were removed and the medium was changed for a new RPMI 1640 medium containing 10% heat-inactivated FBS, the mixture of L‑glutamine and GlutaMax, and the antibiotics/antimycotics. For differentiation of monocytes into macrophages, cultures were maintained for 7 to 10 days in a humid atmosphere at 39°C with 5% CO2, with the medium being refreshed every 2 to 3 days. Once the cell displayed classical macrophage morphology, the medium was changed for complete media without the antibiotics/antimycotics. The macrophages were then incubated overnight under the same conditions for the MAP infection experiment on the following day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was extracted using the RNeasy kit (Qiagen) with the on-column DNase treatment according to the manufacturer’s recommendations. The quality of the 72 samples (four infection and two control time points per animal) was assessed using the Bioanalyzer RNA 6000 kit.
Following rRNA removal using the Ribo-Zero Gold kit (Illumina, Victoria, British Columbia, Canada), 72 cDNA libraries (12 animals × 6 samples, including infection and control time points) were generated from 250 ng of total RNA using the Illumina TruSeq Stranded Total mRNA Sample Preparation kit (Illumina). The length of the fragments was assessed using the Agilent High Sensitivity DNA Chip (Agilent Technologies) on a 2100 Bioanalyzer (Agilent Technologies), and the concentration was determined using a NanoDrop spectrophotometer. Libraries were sequenced at the McGill University and Génome Québec Innovation Centre (MUGQIC) (McGill University, Montreal, Quebec, Canada). Samples were multiplexed in equal ratios (six libraries per two sequencing lanes, equivalent to three libraries per lane) and sequenced in the form of 50-cycle 100 bp paired-end reads on a HiSeq 2000 instrument (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The analysis was performed on the MP2 computing cluster of Compute Canada at the Université de Sherbrooke using the RNA-seq pipeline v.1.2 developed by the McGill University and Genome Québec Innovaction center (MUGQIC) team
Trimming and clipping were done using Trimmomatic software v0.32. Trimming was performed by removing extremities that had a phred quality score below 20 in each four-nucleotide window. The same quality pass or fail criteria were also used for the entire read.
Reads with a minimum length of 32 nucleotides were aligned using TopHat software v2.0.9 to the Btau 4.6.1 reference genome , allowing for two mismatches per read. The quality of the alignment was verified by checking for high levels of duplicated reads and low transcript coverage with the RSeQC package.
The FPKM calculated by Cufflinks software v2.1.1 was used to determine the transcript expression rate.
Genome_build: Btau 4.6.1
Supplementary_files_format_and_content: All processed data files are the unmodified cufflinks output file that calculted the FPKM for each genes (genes.fpkm_tracking) renamed using the sample names
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| Submission date |
Apr 29, 2017 |
| Last update date |
Dec 07, 2019 |
| Contact name |
Nathalie Bissonnette |
| E-mail(s) |
nathalie.bissonnette@agr.gc.ca
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| Phone |
1(819)780-7253
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| Organization name |
Agriculture and Agri-food Canada
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| Department |
Sherbrooke research center
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| Street address |
2000 rue College
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| City |
Sherbrooke |
| State/province |
Quebec |
| ZIP/Postal code |
J1M 0C8 |
| Country |
Canada |
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| Platform ID |
GPL15749 |
| Series (1) |
| GSE98363 |
Mycobacterium avium ssp. paratuberculosis Infection Induces an Immune Tolerance Phenotype in Primary Bovine Macrophages from Johne’s Disease Cows (Agriculture and Agri-Food Canada_N.Bissonnette_J000079) |
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| Relations |
| BioSample |
SAMN06849253 |
| SRA |
SRX2770290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2592725_D_CTL24_genes_fpkm_tracking.tsv.gz |
452.7 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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