|
| Status |
Public on Dec 02, 2019 |
| Title |
HC9_12hrs_Msmeg |
| Sample type |
SRA |
| |
|
| Source name |
Peripheral Blood Mononuclear Cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral Blood Mononuclear Cells
infection: Infected with M Smeg
time point: 12 hours post infection
|
| Treatment protocol |
Mycobacteria was cultured in 7H9 (added 10% OADC and 0.5% glycerol) without Tween. Human macrophages were infected with each mycobacterial strain at MOI=1 and permitted for four hours of uptake. The cells were washed three times with phosphate buffered saline (PBS). Cells were then incubated for another 8 (12 hours post infection) or 92 hours (96 hours post infection) in 5% CO2 incubator at 37C.
|
| Growth protocol |
Phlebotomy was performed on the healthy individuals (80 ml blood) in Na-Heparin vacutainers after obtaining written informed consent. The whole blood was further mixed with PBS in 1:1 ratio. This diluted mixture was gently poured over the Histopaque layer and subjected to centrifugation at 1440 g for 20 min. The buffy coat layer that appears between the blood plasma and the RBCs, containing PBMCs was collected in different tubes and washed twice with PBS. Total cells obtained were counted using a cell Countess (Invitrogen, Company, Country) and validated through manual counting. Human macrophage-like cells were cultured from human blood in Teflon jars using RPMI-1640 medium supplemented with 20% heparinized plasma and incubated at 370C, 5% CO2 for 5 days. This allows the differentiation of monocytes into macrophages
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from human macrophages was extracted with the help of a kit (RNeasy Plus Mini Kit) as per the manufacturer’s instructions. The extraction was performed immediately following the 12- and 96-hours infection period. The ‘gDNA eliminator’ column included in the kit was used to remove genomic DNA in all samples. For each experiment, RNA quantity and quality was measured using Agilent 2100 Bioanalyzer. The RNA with a high RNA integrity Number (RIN) (≥ 9) was used for Ampliseq and quantitative real time qPCR experiments. Total RNA was extracted and frozen immediately at -800C until Ampliseq is performed
Total RNA (100ng) was reverced transcribed using the SuperScript® VILO™ cDNA Kit to generate cDNA. The target regions were amplified for 10 reaction cycles using the Human Gene Expression Core Panel. After partial digestion, barcode adaptors (from the IonCde Adapters Kit) were ligated and the libraries were purified with Agencourt ™ AMPure™ XP reagent and eluted in low TE buffer
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
total RNA targeted and seqeunced using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit
|
| Data processing |
Basecalling by Torrent Suite V5.4
Read mapping by tmap V5.4 to aligned to hg19 AmpliSeq Transcriptome 21 K
reads shorter than 8 filtered out, Quality trimmed to QV>16 with 30bp moving window.
Quantified to transcriptome in Partek (EM method)
Differential expression, (Gene Specific Analysis- Partek), read count of 0 reset to 1.0E-4
Genome_build: hg19 AmpliSeq Transcriptome 21 K
|
| |
|
| Submission date |
Nov 16, 2018 |
| Last update date |
Dec 02, 2019 |
| Contact name |
Abhilasha Madhvi |
| Organization name |
Stellenbosch University
|
| Street address |
Francie van Zijl Drive. Tygerberg
|
| City |
Bellville |
| State/province |
Western Cape |
| ZIP/Postal code |
7505 |
| Country |
South Africa |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE122619 |
Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria |
|
| Relations |
| BioSample |
SAMN10435256 |
| SRA |
SRX5017403 |
