|
| Status |
Public on Jun 01, 2015 |
| Title |
DC_MTB_rep5 (RNA-Seq) |
| Sample type |
SRA |
| |
|
| Source name |
Monocyte-derived dendritic cells
|
| Organism |
Homo sapiens |
| Characteristics |
condition: MTB-infected
|
| Treatment protocol |
Dendritic cells (DCs) were infected with a Mycobacterium tuberculosis (MTB) strain expressing green-fluorescent protein (H37Rv) for 18 h at a multiplicity of infection of 1-to-1. Full details can be found in Barreiro et al. (2012).
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll-Paque centrifugation. Blood monocytes were then purified from PBMCs by positive selection with magnetic CD14 MicroBeads (Miltenyi Biotech). Pure monocytes were cultured for 5 days in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS (Dutscher), L-glutamine (Invitrogen), GM-CSF (20 ng/mL; Immunotools), and IL-4 (20 ng/mL; Immunotools). Cell cultures were fed every 2 days with complete medium supplemented with the cytokines previously mentioned. Before infection, we systematically checked the differentiation/activation status of the monocyte-derived DCs by flow cytometry, using antibodies against CD1a, CD14, CD83, and HLA-DR. Only samples presenting the expected phenotype for non-activated DCs – CD1a+, CD14-, CD83-, and HLA-DRlow – were used in downstream experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the same samples using the miRNeasy kit (Qiagen). RNA quantity was evaluated spectrophotometrically, and the quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies). Only samples with no evidence for RNA degradation (RNA integrity number > 8) were kept for further experiments.
RNA-Seq libraries for the six samples for which we collected MethylC-Seq were generated via polyA+ selection of mRNA from total RNA using the TruSeq RNA Sample Prep Kit v2 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Adaptor sequences and low quality score bases were trimmed and resulting reads were aligned to the human genome reference sequence (GRCh37/hg19).
The number of read fragments overlapping with annotated exons of genes was tabulated using HTSeq using the following parameters: -q -m intersection-nonempty -s no.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include expression count values for each sample.
|
| |
|
| Submission date |
Dec 15, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alain Pacis |
| E-mail(s) |
alain.pacis@mcgill.ca
|
| Organization name |
Canadian Centre for Computational Genomics (C3G)
|
| Street address |
740 Dr Penfield Ave
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3A 0G1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE64179 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells (mRNA-Seq) |
| GSE64183 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells |
|
| Relations |
| BioSample |
SAMN03268991 |
| SRA |
SRX818249 |
