|
Status |
Public on Jan 01, 2020 |
Title |
Uninfected_Donor4_Untreated |
Sample type |
SRA |
|
|
Source name |
Blood mononuclear cells
|
Organism |
Homo sapiens |
Characteristics |
donor: 4 cell type: human monocyte-derived macrophages infection status: uninfected treatment status: Untreated
|
Treatment protocol |
Macrophages from 4 donors were left untreated (control) or treated with 0.5 (BDQ1) or 5 (BDQ2) µg/mL bedaquiline for 18 h. Concerning infected cells, M. tuberculosis H37Rv was grown from a frozen stock to mid-log phase in 7H9 medium (Becton Dickinson), supplemented with albumin-dextrose-catalase (ADC, Difco). Before infection, bacteria were washed three times and re-suspended in 1 ml PBS. Clumps were dissociated by 30 passages through a needle and then allowed to sediment for 5 min. The density of bacteria in the supernatant was verified by measuring the OD600 and aliquot volumes defined to allow one bacterium-per two cell infections. Cells were infected in 6-well plates with each well containing 1.3 × 106 cells in 3 ml medium containing GM-CSF (R&D Systems). After 2 h of incubation at 37°C, infected cells were washed three times in PBS to remove extracellular bacteria and incubated in fresh medium. After 18h, macrophage from 4 donnors were treated with 0.5 (BDQ1) or 5 (BDQ2) µg/mL bedaquiline for 18 h. After incubation, the cell transcriptome was analyzed by mRNA sequencing.
|
Growth protocol |
Blood mononuclear cells were isolated by Ficoll-Paque centrifugation (GE Healthcare Life Sciences). CD14+ monocytes were isolated by positive selection using CD14 microbeads (Miltenyi Biotec) and allowed to differentiate into Mφs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL; R&D Systems) over a six-day period. RPMI medium + GM-CSF was added to the cell cultures every two days.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA from macrophages was extracted using QIAzol reagent (Life Technologies) and purified over RNeasy columns (Qiagen). The quality of all samples was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies) to verify RNA integrity. Only samples with a good RNA yield and no RNA degradation (ratio of 28S to 18S, > 1.7; RNA integrity number > 9) were used for further experiments. cDNA libraries were prepared with the Illumina TruSeq RNA Sample Preparation Kit v2 and were sequenced on an llumina HiSeq 2500
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
STAR v2.5.0b was used to map RNA-seq reads to the hg38 reference genome and quantify gene expression (option--quantMode GeneCounts) by counting the fragments overlapping the Ensembl genes (GRCh38 v. 83). Genome_build: hg38 Supplementary_files_format_and_content: The raw counts were generated using STAR v2.5.0b for GRCh38 Ensembl genes
|
|
|
Submission date |
Jun 21, 2019 |
Last update date |
Jan 02, 2020 |
Contact name |
Alexandre Giraud-Gatineau |
E-mail(s) |
alexandre.giraud.gatineau@gmail.com
|
Organization name |
Institut Pasteur
|
Department |
Microbiology
|
Lab |
integrated mycobacterial pathogenomics
|
Street address |
25-28 rue du docteur roux
|
City |
Paris |
ZIP/Postal code |
75015 |
Country |
France |
|
|
Platform ID |
GPL16791 |
Series (1) |
GSE133145 |
Bedaquiline remodels the macrophage response |
|
Relations |
BioSample |
SAMN12107477 |
SRA |
SRX6102469 |