|
| Status |
Public on Sep 30, 2017 |
| Title |
489_017 |
| Sample type |
SRA |
| |
|
| Source name |
bovine monocyte-derived macrophage
|
| Organism |
Bos taurus |
| Characteristics |
animal identifier: 2721
infection: Mycobacterium bovis strain AF2122/97
time post infection: 24
|
| Treatment protocol |
Frozen mycobacteria stocks were thawed and prepared in medium and inoculated at a multiplicity of infection (MOI) of 5. Medium only was added to the uninfected controls. Flasks containing bMDM were rocked every 15 minutes for two hours and the medium was replaced six hours post infection.
|
| Growth protocol |
Generation of monocyte-derived macrophages - Peripheral blood was asceptically collected from the jugular vein into anticoagulant and PBMC were separated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Life Sciences). The resulting PBMC were cultured at 9 x 107 cells per 75 cm3 flask in Mø medium [RPMI-1640 medium (Invitrogen) supplemented with 15% foetal bovine serum (Lonza Biologicals), 20 mM L-Glutamine, non-essential amino acids (Invitrogen), 50 mM HEPES and antibiotics/antimycotics (Invitrogen)]. After 2-3 hours culture at 37ºC and 5% CO2, the non-adherent cells were removed and the adherent cells cultured for 9 days in Mø medium. The Mø medium was replaced after 24h and then after every 48-72 hours. The bMDM were detached using cell dissociation solution (Sigma-Aldrich) and replated on day 9, which was found to promote differentiation and improve the homogeneity of the bMDM. On approximately day 13, the bMDM were harvested as described above and transferred to 25 cm3 flasks, at up to 3 x 105 cells/flask and cultured for a further 24 hours before infection.
Generation of Mycobacteria stocks - The isolates were cloned prior to growth to mid-log phase in liquid Middlebrook 7H9 medium supplemented with 10% albumin-dextrose-catalase (ADC). The mycobacteria were then harvested and prepared as single cell cultures and frozen at 1 x 107 colony forming units (CFU)/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the desired time point the cells were lysed with 2.5 ml Trizol reagent (Invitrogen) for 20 minutes, to ensure mycobacterial inactivation. RNA was extracted from the samples following the manufacturer's instructions.
mRNA was purified from the total RNA and fractionated to generate fragments with a medium size of 180-210 nucleotides. These fragments were reverse transcribed using random primers to generate cDNA libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
Reads were aligned to the Bovine genome (UMD 3.1) (Ensembl release 63) using the TopHat aligner (ver 1.2.0) with settings -r 300 --mate-std-dev 100 --solexa1.3-quals -G cow.gtf
Data were filtered to only genes with at least 1 count per million reads
Genome_build: Bovine genome (UMD 3.1) from Ensembl release 63
Supplementary_files_format_and_content: CSV files generated using edgeR. Gene counts were filtered to remove genes with zero counts across all samples and with less than one count per million in line with guidelines published by the edgeR authors. The dataset was then normalised using the trimmed mean normalisation (Robinson and Oshlack 2010).
|
| |
|
| Submission date |
Sep 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Iain Gallagher |
| E-mail(s) |
iaingallagher@gmail.com
|
| Phone |
00 44 1786 46 6024
|
| Organization name |
University of Stirling
|
| Department |
Faculty of Health Sciences and Sport
|
| Street address |
Room 4B133 Cottrell Building, University of Stirling, Airthrey Road
|
| City |
Stirling |
| ZIP/Postal code |
FK9 4LA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11153 |
| Series (1) |
| GSE104211 |
Transcriptional response of bovine moncocyte-derived macrophages to infection with strains of Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis |
|
| Relations |
| BioSample |
SAMN07693460 |
| SRA |
SRX3213605 |
