|
| Status |
Public on Mar 31, 2015 |
| Title |
M373-Rv+-18 |
| Sample type |
SRA |
| |
|
| Source name |
monocyte-derived macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
individual: M373
agent: Rv+
time point: 18
batch: 7
rin: 9.3
illumina_index: 13
|
| Treatment protocol |
For each bacterial infection, we treated the macrophages with a multiplicity of infection (MOI) of 2:1. After one hour, we washed the macrophages five times with phosphate-buffered saline (PBS) and treated them with gentamycin (50 µg/µL) to kill all extracellular bacteria. After one hour of antibiotic treatment, we changed the medium to a lower concentration of gentamycin (5 µg/µL), which marked the zero timepoint of the study. We allowed the cells to grow for 4, 18, or 48 hours before lysing them before lysing them with QIAzol Lysis Reagent and then storing them at -80° C.
|
| Growth protocol |
First, we collected buffy coats (~50 mL). Next we isolated peripheral blood mononuclear cells (PBMCs) via Ficoll-Paque centrifugation nd enriched for monocytes via positive selection with beads containing CD14 antibodies. Then we differentiated the monocytes into macrophages by culturing for 6-7 days in RPMI buffer supplemented with macrophage colony-stimulating factor (M-CSF).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We extracted RNA using the QIAgen miRNeasy kit. There were a total of 13 batches of 12 samples each (6 individuals x 9 conditions x 3 timepoints, minus 48 hours post-infection with Staphylococcus epidermidis). We designed the batches to maximally partition the variables of interest (individual, condition, timepoint) in order to minimize the introduction of biases due to batch processing.
In batches of 12 samples (the same 13 from the extraction protocol), we added barcoded adapters (Illumina TruSeq RNA Sample Preparation Kit v2)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
M373.Rv+.18
|
| Data processing |
We mapped the short reads to the human genome (hg19) using Subread (default parameters).
We obtained the read counts for each Ensembl protein-coding gene (biotype: “protein_coding”) with featureCounts, which sums the reads falling in the union of all exons of a gene and discards reads mapping to more than one gene (default parameters, except that we provided the Ensembl annotation file as described).
We removed genes with fewer than one count per million exonic reads in fewer than six samples.
We normalized the samples using the weighted trimmed mean of M-values algorithm (TMM) in edgeR.
We converted the counts to log counts per million using the cpm function in edgeR (default parameters).
We removed the effect of RNA quality (RIN) score and extraction batch using removeBatchEffect from limma.
Genome_build: hg19
Supplementary_files_format_and_content: table-s1.txt is a tab-delimited text file that contains the batch-corrected log2 counts per million for each of the 156 samples, as well as the Ensembl gene ID and gene name.
|
| |
|
| Submission date |
Mar 31, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
John D Blischak |
| Organization name |
University of Chicago
|
| Department |
Human Genetics
|
| Lab |
Gilad
|
| Street address |
920 E. 58th Street, CLSC 317
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60615 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE67427 |
Mycobacterial infection induces a specific human innate immune response |
|
| Relations |
| BioSample |
SAMN03452441 |
| SRA |
SRX974137 |
